Fluorescent compounds specific for pluripotent stem cells and reprogramming-ready cells and methods of using the same

ABSTRACT

Determining, in a sample, the presence and/or amount of pluripotent stem cells. A sample suspected of containing one or more pluripotent stem cells is provided. The sample with a fluorescent compound is contacted under conditions that allow binding of the fluorescent compound to the pluripotent stem cells, where the fluorescent compound is a fluorescent compound of formula (I) or a pharmaceutically acceptable salt thereof. The presence and/or amount of the pluripotent stem cells is determined by measuring the fluorescence of the cells following the contacting.

Pursuant to 37 C.F.R. § 1.834, Applicant has submitted a sequencelisting as an XML file (“Sequence Listing”). The name of the filecontaining the Sequence Listing is“40940-P162WOUSD1_SequenceListing.xml.” The date of the creation of theSequence Listing is Mar. 11, 2023. The size of the Sequence Listing is1,345 kilobytes. Applicant hereby incorporates by reference the materialin the Sequence Listing.

FIELD OF THE INVENTION

The present invention relates generally to compounds specific forpluripotent stem cells and reprogramming cells and the methods of usingthe same.

BACKGROUND OF THE INVENTION

The discovery of human induced pluripotent stem (iPS) cells hasrevolutionized and accelerated the new development of personalized drugscreening, human disease modeling, and regenerative therapeutics.Despite rapid development of methods to derive human iPS cells, therehave been several problems and challenges with the reprogrammingprotocols. These include relatively low efficiency of obtaining highquality cells, long duration of complete reprogramming processes(typically 3-4 weeks before colony formation), and difficulty in promptanalysis and identification of high quality iPS cells. The lowefficiency and long time-course worsen when clinically applicableprotocols are attempted by adapting non-viral transduction andfeeder-free reprogramming methods. Efficiency and time can be improvedusing selective cell types. For example, the inventors of the presentapplication previously found that adipose-derived stem cells (ASCs) anddental pulp-derived stem cells (DPSCs) allow feeder-free reprogrammingwith relatively high efficiencies and shorter time frames.

However, the technology to promptly distinguish bona fide pluripotentstem cells from other somatic cell populations is still underdeveloped.Traditionally, gene reporters such as fluorescent proteins driven underOCT4, NANOG, or artificial reporters have been used. However, theseconstructs need to be inserted into cells by viral, gene editing orother genetic engineering methods and successful expression verified,which are cumbersome and potentially disruptive to endogenous genomefunction and are not widely applicable for diverse ranges of cell types.

Using fluorescent dye conjugated antibodies for pluripotent cell surfacemarkers such as TRA-1-60/81 and SSEA3/4, or fluorescent substrates foralkaline phosphatase is the most common method to detect iPS cells(Quintanilla, et al., (2016). J Vis Exp.). However, alkaline phosphataseand SSEA3/4 are not very specific to bona fide pluripotent stem cells,and are detectable in adult stem cells including adipose-derived stromalcells (ASCs) and dental pulp stem cells (DPSCs). All these markerstypically stain well-developed colonies of iPS cells only, which can bevisible and recognized by experienced observers even under phasecontrast microscopy. In addition, it is relatively expensive tomanufacture these fluorescent probes, which may hinder the clinical andcommercial development of iPS technology.

KP-1 is a fluorescent probe that is reportedly specific for human iPScells. However, it fails to enable the sorting of early reprogrammingcells to enrich colony-forming iPS cells (Hirata et al., 2014). Theinventors previously identified CDy1, a small-molecule fluorescentprobe, by screening against mouse ES and iPS cells (Im, et al. (2010).Angew Chem Int Ed Engl 49, 7497-7500; Kang, et al. (2011). Nat Protoc 6,1044-1052). CDy1 allowed early stage live cell staining and sorting ofreprogramming cells.

However, there remains a considerable need for new fluorescent probesthat specifically detect iPS cells at an early reprogramming stage.

SUMMARY OF THE INVENTION

The inventors of the present invention have found that said need can bemet by the provision of the fluorescent compounds disclosed herein.

In a first aspect, the present invention relates to a fluorescentcompound of formula (I) or a pharmaceutically acceptable salt thereof

wherein:

-   -   R₁, R₄, R₅, R₈, R₉ and R₁₀ are each independently selected from        the group consisting of H and C₁₋₆ alkyl;    -   R₂, R₃, R₆ and R₇ are each independently selected from C₁₋₆        alkyl;    -   R₁₁ is selected from the group consisting of H,        (CR₁₂R₁₃)_(o)—N(R₁₄R₁₅) and C₁₋₆ alkyl, wherein o is        independently 0, 1, 2, 3, 4, or 5, and R₁₂-R₁₅ are each        independently selected from the group consisting of H and C₁₋₆        alkyl;    -   m and n are each independently 0, 1, or 2;    -   p is 0, 1, 2, or 3,    -   q is 1, 2, 3, or 4, with the proviso that p+q≤4; and    -   means that the respective bond can be a single or double bond        and, if it is a single bond, the additional valencies are        hydrogen.

In various embodiments, R₁, R₄, R₅, R₈, R₉ and R₁₀ are eachindependently selected from the group consisting of hydrogen and methyland/or R₂, R₃, R₆ and R₇ are each methyl and/or R₁₁ is (CH₂)₂—N(CH₃)₂.

In preferred embodiments, the fluorescent compound is a compound offormula (II)

In a second aspect, the invention relates to a method of determining, ina sample, the presence and/or amount of pluripotent stem cells, saidmethod comprising the steps of:

-   -   (i) providing a sample suspected of containing one or more        pluripotent stem cells;    -   (ii) contacting the sample with a fluorescent compound disclosed        herein under conditions that allow binding of said fluorescent        compound to the pluripotent stem cells; and    -   (iii) determining the presence and/or amount of the pluripotent        stem cells by measuring the fluorescence of the cells following        said contacting.

In various embodiments, the pluripotent stem cells are embryonic stemcells or induced pluripotent stem cells.

In various embodiments, the pluripotent stem cells are mammalian cells,preferably human, mouse, or rat cells, more preferably human cells.

In various embodiments, step (ii) does not comprise a washing stepfollowing said contacting.

In various embodiments, the method further comprises a step of:

-   -   (iv) isolating the pluripotent stem cells labelled by the        fluorescent compound from the sample.

In various embodiments, the labelled cells are isolated byfluorescence-activated cell sorting (FACS).

In a third aspect, the invention relates to a method of determining, ina sample, the presence and/or amount of cells undergoing reprogrammingto become induced pluripotent stem cells, said method comprising thesteps of:

-   -   (i) providing a sample suspected of containing one or more cells        undergoing reprogramming to become induced pluripotent stem        cells;    -   (ii) contacting the sample with a fluorescent compound disclosed        herein under conditions that allow binding of said fluorescent        compound to the cells undergoing reprogramming to become induced        pluripotent stem cells; and    -   (iii) determining the presence and/or amount of the cells        undergoing reprogramming to become induced pluripotent stem        cells by measuring the fluorescence of the cells following said        contacting.

In various embodiments, the cells undergoing reprogramming to becomeinduced pluripotent stem cells are mammalian cells, preferably human,mouse, or rat cells, more preferably human cells.

In various embodiments, step (ii) does not comprise a washing stepfollowing said contacting.

In various embodiments, the method further comprises a step of:

-   -   (iv) isolating the cells undergoing reprogramming to become        induced pluripotent stem cells labelled by the fluorescent        compound from the sample.

In various embodiments, the labelled cells are isolated byfluorescence-activated cell sorting (FACS).

In a fourth aspect, the invention relates to the use of the fluorescentcompound disclosed herein in the detection and/or isolation ofpluripotent stem cells.

In another aspect, the invention relates to the use of the fluorescentcompound disclosed herein in the detection and/or isolation of cellsundergoing reprogramming to become induced pluripotent stem cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood with reference to the detaileddescription when considered in conjunction with the non-limitingexamples and the accompanying drawings.

FIG. 1 : (A) Schematic diagram showing the screening process offluorescent probes for somatic and iPS cells using DOFLA. Cells wereseeded on either MEFs or MG in 384-well plates for primary screening and96-well plates for secondary and tertiary screening. After 48 h, cellswere stained with probes for 1 h and images were taken in theImageXpress under various conditions as indicated. (B) The chemicalstructure of BDL-E5.

FIG. 2 : (A) Fluorescent images (10×objective) of BDL-E5 probe (No wash)and Hoechst 33342 staining of AiPS1 colonies and its original ASC1 (ASCline #1) on MEF- (i) and MG-coated (ii) plates from primary screening(n=3). (B) Fluorescent images (10×) of BDL-E5 probe (No wash), Hoechstand TRA-1-60 on DiPS1 colonies and DPSC1 (DPSC line #1) on MEF- andMG-coated plates from secondary screening (n=3). (C) Fluorescent images(10×) and average fluorescence intensity of BDL-E5 probe (No wash) andTRA-1-60 on ASC2 (i), (iii) and DPSC1 (ii), (iv) on MG-coated plates at7, 14, 21, 28 days post nucleofection (dpn) with reprogramming factors.*Represents the same images. Cells were incubated with 500 nM of BDL-E5in appropriate media for 1 h (n=3). Scale bar represents 100 μm.

FIG. 3 : Images showing BDL-E5 staining tracked daily on reprogrammedcells of ASC4 and DPSC1 on MG. Representative images (10×) were taken at10, 13, 17, 20 and 24 dpn (n=3). Scale bar represents 100 μm.

FIG. 4 : (A) Histogram (FACS) showing BDL-E5+ cell populations at 7 dpn:DPSC1 (i) and DPSC2 (ii). Relative cell count is indicated on they-axis, and fluorescence intensity (Texas Red channel) on the x-axis.The top 10% and bottom 10% of cell populations are as indicated (n=3).(B) (i) Fluorescent images of BDL-E5, TRA-1-60 and transmitted light(TL) images (4×) showing iPS colonies derived from BDL-E5+ and BDL-E5⁻cell populations of DPSC1 and DPSC2 following FACS at 7 dpn. (ii) Graphshowing average number of DiPS colonies in reprogrammed BDL-E5+ andBDL-E5⁻ cell populations obtained after FACS of DPSC1 and DPSC2.**p<0.01 and ***p<0.001 denote statistical significance (n=3). (C)Histogram showing FACS of BDL-E5+ and BDL-E5⁻ cell populations at 14 dpnfrom SC-ASC S15 (i) and VS-ASC S15 (ii). The percentage of positivelyand negatively stained cells is shown. (iii) Graph showing averagenumber of iPS colonies in BDL-E5+ and BDL-E5⁻ cell populations obtainedafter FACS at 14 dpn from SC-ASC S15 and VS-ASC S15. *p<0.05 denotesstatistical significance. (iv) TL images (10×) showing iPS colonies fromSC-ASC S15 and VS-ASC S15 following FACS for BDL-E5+ and BDL-E5⁻ cellpopulations. Similar results were obtained with S16-derived SC-ASC andVS-ASC (n=3). Scale bar represents 100 μm.

FIG. 5 : Representative graphs showing gene expression of DNMT3B, GDF3,and Nanog (A); LIN28 (B); DPPA2 (C); Cdh1, and EpCAM1 (D); ZEB1, ZEB2,Snail1, and Snail2 (E); TGF-β1 (F), FN1 (G); and Activin A (H) in RNAisolated from DiPS2, DPSC2, BDL-E5+, and BDL-E5-cells of DPSC2 obtainedafter FACS at 7 dpn (n=3). DiPS2 colonies were generated from theoriginal DPSC2 cells that were similarly subjected to FACS. *p<0.05 and**p<0.01 denote significance compared with DiPS2; {circumflex over( )}p<0.05 and {circumflex over ( )}{circumflex over ( )}p<0.01 denotesignificance compared with BDL-E5⁺.

FIG. 6 : (A) Heatmap showing 386 differentially expressed genes (betweenBDL-E5⁺ and BDL-E5⁻ cells) after RNA sequencing of four cell types(DiPS, DPSC, BDL-E5⁺ and BDL-E5⁻ cells) after FACS at 7 dpn of DPSC2upon reprogramming (n=2). (B) Venn diagram showing the number of genesup-regulated in BDL-E5⁺ and BDL-E5⁻ cells, as obtained from RNAsequencing data. A total of 386 genes were differentially expressedsignificantly between the two cell types. (C) Table showing the relevantgenes that were differentially expressed among DiPS, DPSC, BDL-E5⁺, andBDL-E5⁻ cells. (D) Graphs showing mRNA expression of CREB1 (i) andPRKAB2 (ii) obtained by qPCR from RNA isolated from DiPS, DPSC, BDL-E5⁺,and BDL-E5⁻ cells of DPSC2 obtained after FACS at 7 dpn (n=3). *p<0.05denotes significance compared with DiPS; {circumflex over ( )}p<0.05 and{circumflex over ( )}{circumflex over ( )}{circumflex over ( )}<0.001denote significance compared with BDL-E5⁺.

FIG. 7 : (A) Graph showing mRNA expression of CREB1 in Scr CREB1, CREB1OE and siCREB1 DPSC1 and ASC1 (n=3). *p<0.05 and **p<0.01 denotesignificance compared with Scr DPSC1/ASC1. (B) TL and fluorescenceimages (10×) of BDL-E5, TRA-1-60 showing PS colonies derived from DPSC1transfected with Scr CREB1, CREB1 OE and siCREB1 at 12 dpn (n=3). Scalebar represents 100 μm. (C) Graph showing the average number of coloniesin reprogrammed DPSC1 and ASC1 transfected with Scr CREB1, CREB1 OE, andsiCREB1 at 12 dpn (n=3). **p<0.01 and ***p<0.001 denote statisticalsignificance.

FIG. 8 : (A) Fluorescent images (10×objective) of CDy1 probe (Wash 180min) and Hoechst on AiPS1 colonies and ASC1 on (i) MEF- and (ii)MG-coated plates from primary screening (n=3). (B) Fluorescent images(10×) of CDy1 probe (Wash 180 min), Hoechst and TRA-1-60 on DiPS1colonies and DPSC1 on MEF- and MG-coated plates from secondaryscreening. Cells were incubated with 500 nM of CDy1 in appropriate mediafor 1 h (n=3). Scale bar represents 100 μm.

FIG. 9 : (A, B) Fluorescent images (10×) of BDL-E5, CDy1 and Hoechst onAiPS1 colonies on MEF- and MG-coated plates at different conditions (Nowash, Wash 0 min, Wash 60 min, Wash 180 min) after incubation with 500nM probe for 1 h (n=3). *Represents the same images that are presentedin FIGS. 2 and 8 . (C) Fluorescent images (10×) of BDL-E5 and CDy1 onAiPS3 colonies on MEF- and MG-coated plates at different conditions (Nowash, Wash 0 min, Wash 60 min) after incubation with 500 nM probe for 1h (n=3). Scale bar represents 100 μm.

FIG. 10 : (A) (i)-(iv) Histogram (FACS) showing unstained populations ofcells used as the control for FACS performed in FIG. 4 . (B)Fluorescence images of BDL-E5, TRA-1-60 and transmitted light (TL)images showing iPS colonies derived from ASC4 14 dpn BDL-E5⁺ (i) andBDL-E5⁻ (ii) cells at passage 0 (4×) and passage 4 (10×) (n=3). Scalebar represents 100 μm. (C) Graph showing average number of iPS coloniesfrom BDL-E5⁺ and BDL-E5⁻ cell populations at 14 dpn in ASC4 at passage 0(n=3). ***p<0.001 denotes statistical significance.

FIG. 11 : (A) Representative graphs showing gene expression of LIN28(i), NANOG (ii), Activin A (iii) and TGF-β1 (iv) in RNA isolated fromAiPS4, ASC4, BDL-E5⁺ and BDL-E5⁻ cells of ASC4 at 14 dpn. *p<0.05 and***p<0.001 denote significance compared with AiPS4; {circumflex over( )}{circumflex over ( )}p<0.01 denotes significance compared withBDL-E5⁺ (n=3). (B) (i) Fluorescence images (10×) of TUJ1, SMA, AFP, DAPIand TL of cells following spontaneous differentiation of EBs generatedfrom BDL-E5⁺ DPSC1. (ii)-(v) Representative graphs showing geneexpression of GATA2, SMA, AFP and SOX7 in RNA isolated from DiPS1 andspontaneously differentiated cells from EBs formed from BDL-E5⁺ iPScells. ***p<0.001 and ****p<0.0001 denote significance compared withDiPS1 (n=3). (C) Phase contrast (PC) and fluorescent images of BDL-E5and TRA-1-60 of reprogramming SC-ASC S16 and DPSC2 on Geltrex™-coatedCytodex 3 microcarriers at 14 dpn (10×) and 21 dpn (20×). Scale barrepresents 100 μm. (D) Fluorescent images of reprogramming DPSC2 on MGcoated chamber slides at 7 dpn (n=3). These images are zoomed in andcropped from 20×images to clearly show the stains and their overlap;green—markers for Endoplasmic Reticulum (ER), Golgi, Lysosome, orMitochondria; red—BDL-E5; blue—Hoechst 33342.

FIG. 12 : (A) DPSC1 was reprogrammed with the traditional methodinvolving retroviral OCT4, SOX2, KLF4 and C-MYC, and plated onto the MEFfeeder layer. Cells were co-stained with BDL-E5 and TRA-1-60 in theindicated day post-infection (dpi). (B) BJ fibroblasts were transducedwith lentiviral OCT4, SOX2, KLF4 and C-MYC in the presence or absence ofA83-01 (0.3 μM) and stained at 8 dpi. The image is merged from 9independent fields. (C) BJ fibroblasts transduced above were stainedwith BDL-E5 followed by cell fixation and immunostaining with TRA-1-60at 21 dpi.

FIG. 13 : (A) Pathway analysis using Ingenuity Systems (Qiagen) showsrepresentation of the top networks and canonical pathways betweenBDL-E5⁺ and BDL-E5⁻ cells. The molecular and cellular functions thatwere differentially expressed in BDL-E5⁺ and BDL-E5⁻ cells are alsorepresented, along with the p values. (B) Metascape gene analysis wasperformed on http://metascape.org and the enriched clusters betweenBDL-E5⁺ vs. BDL-E5⁻ cells are represented here.

FIG. 14 : (A) Graph representing signal-to-noise ratios (arbitraryfluorescence units) on comparing reprogramming (RP) versusnon-reprogramming (non-RP) DPSCs (DPSC1) stained with either CDy1 orBDL-E5. The fluorescence intensity was measured using ImageJ software.100 cells per field (10×), 10 fields per well, 3 wells per probe weremeasured. ****p<0001 denotes significance between RP and non-RP cells.(B) Proliferation assay of DPSC1 incubated with BDL-E5 (500 nM) for 2 to5 days; represented as number of viable cells per cm² (n=3). (C)Proliferation assay of reprogramming DPSC1, 48 h after transfection withScr CREB1, CREB1 OE or siCREB1; represented as number of viable cellsper cm² (n=3).

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description refers to, by way of illustration,specific details and embodiments in which the invention may bepracticed. These embodiments are described in sufficient detail toenable those skilled in the art to practice the invention. Otherembodiments may be utilized and structural, and logical changes may bemade without departing from the scope of the invention. The variousembodiments are not necessarily mutually exclusive, as some embodimentscan be combined with one or more other embodiments to form newembodiments.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. The singular terms “a,” “an,” and “the” include pluralreferents unless context clearly indicates otherwise. Similarly, theword “or” is intended to include “and” unless the context clearlyindicates otherwise. The term “comprises” means “includes.” In case ofconflict, the present specification, including explanations of terms,will control.

In a first aspect, the present invention relates to a fluorescentcompound of formula (I) or a pharmaceutically acceptable salt thereof

wherein:

-   -   R₁, R₄, R₅, R₈, R₉ and R₁₀ are each independently selected from        the group consisting of H and C₁₋₆ alkyl;    -   R₂, R₃, R₆ and R₇ are each independently selected from C₁₋₆        alkyl;    -   R₁₁ is selected from the group consisting of H,        (CR₁₂R₁₃)_(o)—N(R₁₄R₁₅) and C₁₋₆ alkyl, wherein o is        independently 0, 1, 2, 3, 4, or 5, and R₁₂-R₁₅ are each        independently selected from the group consisting of H and C₁₋₆        alkyl;    -   m and n are each independently 0, 1, or 2;    -   p is 0, 1, 2, or 3,    -   q is 1, 2, 3, or 4, with the proviso that p+q≤4; and    -   means that the respective bond can be a single or double bond        and, if it is a single bond, the additional valencies are        hydrogen.

The term “fluorescent compound” as used herein refers to a compoundhaving a functional group or moiety which will molecularly absorbphotonic energy of a specific UV wavelength and subsequently re-emit ofat least a portion of the absorbed energy as photonic energy at adifferent wavelength within the visible light range, i.e. 380 to 700 nm.

Determining whether a compound falling within formula (I) is afluorescent compound is within the knowledge of the person of averageskill in the art. For example, fluorescence microscopy, fluorescencespectrometry, and flow cytometry can be used to detect a signal emittedby a fluorescent compound of formula (I).

The term “alkyl” as used herein refers to a linear, branched, or cyclicsaturated hydrocarbon group. The term “C₁₋₆ alkyl” indicates that thegroup may have from 1 to 6 (inclusive) carbon atoms in it.Representative alkyl groups include methyl, ethyl, n-propyl, i-propyl,n-butyl, t-butyl, n-pentyl, and 3-pentyl and the like.

The term “pharmaceutically acceptable salt” as used herein refers tothose salts that are within the scope of proper medicinal assessment,suitable for use in contact with human tissues and organs and those oflower animals, without undue toxicity, irritation, allergic response orsimilar and are consistent with a reasonable benefit/risk ratio.Pharmaceutically acceptable salts are technically well known.

In various embodiments, R₁, R₄, R₅, R₈, R₉ and R₁₀ are eachindependently selected from the group consisting of hydrogen and methyland/or R₂, R₃, R₆ and R₇ are each methyl and/or R₁₁ is (CH₂)₂—N(CH₃)₂.

In preferred embodiments, the fluorescent compound is a compound offormula (II)

which is referred to herein as BDL-E5.

Without wishing to be bound to any theory, it is believed that thefluorescent compounds of formulae (I) and (II) are non-toxic andspecifically bind to pluripotent stem cells or cells undergoingreprogramming that have been determined to become induced pluripotentstem cells.

As would be readily appreciated by the skilled person, by the term“specific binding” or “specifically bind” is meant that the fluorescentcompounds of this disclosure bind to pluripotent stem cells or cellsundergoing reprogramming to become induced pluripotent stem cells with asubstantially higher selectivity than to non-target cells such assomatic cells, providing for a distinguishing fluorescence signal.Typically, a fluorescence signal indicating the presence and/or amountof said cells is substantially greater than background signal. Forexample, said fluorescence signal can be at least two-fold greater thanthe intensity of background fluorescence signal. Preferably, theintensity of the fluorescence signal is at least five-fold, at leastten-fold, and, most preferably, at least fifty-fold greater than theintensity of background fluorescence signal.

The compounds of formula (I) or (II) disclosed herein may be preparedusing standard techniques known to those skilled in the art of syntheticorganic chemistry, or may be deduced by reference to the pertinentliterature, for example, WO2014109713A1, which discloses a syntheticscheme of BODIPY compounds and is hereby incorporated by reference inits entirety.

In a second aspect, the invention relates to a method of determining, ina sample, the presence and/or amount of pluripotent stem cells, saidmethod comprising the steps of:

-   -   (i) providing a sample suspected of containing one or more        pluripotent stem cells;    -   (ii) contacting the sample with a fluorescent compound disclosed        herein under conditions that allow binding of said fluorescent        compound to the pluripotent stem cells; and    -   (iii) determining the presence and/or amount of the pluripotent        stem cells by measuring the fluorescence of the cells following        said contacting.

The term “determine” refers to any qualitative and/or quantitativeidentification of a subject of interest. “Determining the amount”, asused herein, includes determining the number, such as the absolutenumber, of pluripotent stem cells in a sample.

The term “sample” as used herein refers to a biological sample, or asample that comprises at least some biological materials such as cells.The samples of this disclosure may be any samples suspected ofcontaining one or more pluripotent stem cells or cells undergoingreprogramming to become induced pluripotent stem cells, including solidtissue samples, such as bone marrow, and liquid samples, such as cellcultures, whole blood, blood serum, blood plasma, cerebrospinal fluid,central spinal fluid, lymph fluid, cystic fluid, sputum, stool, pleuraleffusion, mucus, pleural fluid, ascitic fluid, amniotic fluid,peritoneal fluid, saliva, bronchial washes and urine. In someembodiments, the sample is a cell culture grown in vitro. For example,the culture may comprise cells cultured in a culture plate or dish, asuspension of cells, or a 3D culture on microcarriers or in scaffolds.The sample may include a mixed cell population.

The term “pluripotent stem cells” as used herein refers to self-renewingcells that can differentiate into endoderm, ectoderm, and mesodermcells. Pluripotency is also evidenced by the expression of embryonicstem (ES) cell markers. Pluripotent stem cells include, withoutlimitation, embryonic stem cells, induced pluripotent stem cells, andembryonic germ cells. In various embodiments, the pluripotent stem cellsare embryonic stem cells or induced pluripotent stem cells.

Embryonic stem cells originate from the inner cell masses of earlyembryos, and are capable of self-renewal, maintaining pluripotency, anddifferentiating into cells of three germ layers.

Induced pluripotent stem cells are one type of pluripotent stem cellsartificially derived from reprogramming of non-pluripotent cells (e.g.,somatic cells) by, for example, introduction of stem cell pluripotencyfactors, the key factors in maintaining stem cell pluripotency. Somaticcells can be reprogrammed to stem cells by introducing these factorsinto the somatic cells. Many factors have been reported to have anability to induce the reprogramming of somatic cells. Preferably, thepluripotency factor(s) is/are one or more selected from the groupconsisting of Oct4, Sox2 (or Sox1), Klf4 (or Klf2 or KLF5), Nanog, c-Myc(or L-Myc or N-Myc), Lin28 and Esrrb. Said stem cell pluripotencyfactors may be derived from any desired species.

The term “reprogramming” as used herein refers to a process thatreverses the developmental potential of a cell or population of cells(e.g., a somatic cell). Stated another way, reprogramming refers to aprocess of driving a cell to a state with higher developmentalpotential, i.e., backwards to a less differentiated state. The cell tobe reprogrammed can be either partially or terminally differentiatedprior to reprogramming. In the context of the present application,reprogramming encompasses a complete reversion of the differentiationstate, i.e., an increase in the developmental potential of a cell tothat of a cell having a pluripotent state. In some embodiments,reprogramming encompasses driving a somatic cell to a pluripotent state,such that the cell has the developmental potential of a pluripotent stemcell. In some embodiments, reprogramming encompasses driving an adultstem cell to a pluripotent state, such that the cell has thedevelopmental potential of a pluripotent stem cell.

In general, it has been accepted in the art that the induced pluripotentstem cells are equivalent to embryonic stem cells, given that theinduced pluripotent stem cells have the characteristics of: (a) stemcell gene and protein expression; (b) chromosome methylation; (c)doubling time; (d) embryo formation; (e) teratoma formation; (f) viablechimera formation; (g) hybridoma; and (h) differentiation.

The samples and/or the cells disclosed herein may be obtained from anyorganism, including mammals such as humans, primates (e.g., monkeys,chimpanzees, orangutans, and gorillas), cats, dogs, rabbits, farmanimals (e.g., cows, horses, goats, sheep, pigs), and rodents (e.g.,mice, rats, hamsters, and guinea pigs), preferably from humans. Theorganism may be a healthy organism or suffer from a disease condition.Disease conditions may include any disease, such as cancer, diabetes,metabolic syndrome, or an autoimmune disorder.

In accordance with the present invention, a sample suspected ofcontaining pluripotent stem cells is provided, and the pluripotent stemcells comprised in the sample are contacted with and consequentlyspecifically labelled by a fluorescent compound disclosed herein,enabling the subsequent detection and/or measurement of the labelledcells.

In various embodiments, step (ii) does not comprise a washing stepfollowing said contacting.

By virtue of the selectivity of the fluorescent compounds forpluripotent stem cells, following the labeling of step (iii), anadditional step of removing unbound fluorescent labels may be disposedof.

In various embodiments, the method further comprises a step of:

-   -   (iv) isolating the pluripotent stem cells labelled by the        fluorescent compound from the sample.

The compounds disclosed herein, BDL-E5 for example, are not toxic,rendering them suitable for use in isolating labeled living cells fromthose that are not labeled.

In various embodiments, the labelled cells are isolated byfluorescence-activated cell sorting (FACS).

It should be noted that the fluorescent compounds disclosed herein may,in the context of the present application, be used in combination withone or more other detectable labels (e.g. a fluorescently labeledantibody against a cell surface marker and/or a vitality stain such aspropidium iodide), such that a plurality of parameters could bedetermined simultaneously for reliable analysis and/or isolation of thetarget cells.

It is postulated that tumor-initiating cells (also known as cancer stemcells), which reportedly possess some properties of pluripotent stemcells, may also be specifically stained by the compounds of thisdisclosure such as BDL-E5. The identification, characterization, and/orisolation of tumor-initiating cells by said compounds are therefore alsocontemplated to be within the scope of the present invention.

In a third aspect, the invention relates to a method of determining, ina sample, the presence and/or amount of cells undergoing reprogrammingto become induced pluripotent stem cells, said method comprising thesteps of:

-   -   (i) providing a sample suspected of containing one or more cells        undergoing reprogramming to become induced pluripotent stem        cells;    -   (ii) contacting the sample with a fluorescent compound disclosed        herein under conditions that allow binding of said fluorescent        compound to the cells undergoing reprogramming to become induced        pluripotent stem cells; and    -   (iii) determining the presence and/or amount of the cells        undergoing reprogramming to become induced pluripotent stem        cells by measuring the fluorescence of the cells following said        contacting.

Without wishing to be bound to any theory, it is believed that thefluorescent compounds disclosed herein specifically bind to not onlypluripotent stem cells but also cells undergoing reprogramming that havebeen determined to become induced pluripotent stem cells, even at anearly stage of the reprogramming.

For example, the inventors surprisingly found that the fluorescentcompound BDL-E5 specifically stains early reprogramming cells, but notsomatic cells or adult stem cells such as ASCs or DPSCs. In addition, asdetailed below, BDL-E5 allows early detection and enrichment ofreprogramming cells, as early as seven days before stem cell coloniesare visible.

Consistently, BDL-E5⁺ reprogrammed cells exhibit high expression levelsof pluripotent genes, some of which are nearly comparable to those inmature induced pluripotent stem cells. BDL-E5 therefore offers avaluable tool for detecting authentic early reprogramming cells, allowsenrichment of the reprogramming-ready cell population, and helps uncovernovel regulators of reprogramming.

In various embodiments, the cells undergoing reprogramming to becomeinduced pluripotent stem cells are mammalian cells, preferably human,mouse, or rat cells, more preferably human cells.

In various embodiments, step (ii) does not comprise a washing stepfollowing said contacting.

As shown in the Examples of the present application, CDy1 presentedhigher signals toward human reprogramming cells but also higherbackground staining against non-reprogramming cells, but BDL-E5, anexemplary compound of those disclosed herein, exhibited low non-specificstaining against non-reprogramming cells and therefore, unlike CDy1,does not require washing after the staining process to reduce backgroundsignals.

In various embodiments, the method further comprises a step of:

-   -   (iv) isolating the cells undergoing reprogramming to become        induced pluripotent stem cells labelled by the fluorescent        compound from the sample.

In various embodiments, the labelled cells are isolated byfluorescence-activated cell sorting (FACS).

Following isolation, the enriched cells are believed to exhibitproperties of pluripotent stem cells, such as colony formation in vitroand teratoma formation in vivo in immunocompromised recipient animals.

One skilled in the art would readily appreciate that the methoddisclosed herein could also be used to identify agents that inhibit orstimulate cell reprogramming.

In a fourth aspect, the invention relates to the use of the fluorescentcompound disclosed herein in the detection and/or isolation ofpluripotent stem cells.

In another aspect, the invention relates to the use of the fluorescentcompound disclosed herein in the detection and/or isolation of cellsundergoing reprogramming to become induced pluripotent stem cells.

The present invention is further illustrated by the following examples.However, it should be understood, that the invention is not limited tothe exemplified embodiments.

EXAMPLES

Materials and Methods

Isolation of ASCs

WAT was isolated from subcutaneous (abdominal region) and visceral(omental region) depots from 2 human volunteers (S15-S16, undergoingbariatric surgery, with approval by the National Healthcare Group DomainSpecific Review Board at National Healthcare Group, Singapore) asdescribed previously (Ong, et al. (2014). Stem Cell Reports 2, 171-179;Takeda, et al. (2016). Diabetes 65, 1164-1178). S15 is a 24-year oldIndian female and S16 is a 36-year old Indian male. ASCs were isolatedfrom WAT and cultured, as previously described (Sugii, et al. (2011).Nature Protoc 6, 346-358). Cells only up to passage 5 were used forexperiments. MSC cell surface markers and multipotency of ASCs usedherein were confirmed by flow cytometry and differentiation assays,respectively (Ong, et al. (2014). Stem Cell Reports 2, 171-179).

ASC and DPSC Culture

Different lines of ASCs and DPSCs were obtained from commercial sources(Lonza, Invitrogen and PromoCell). ASCs were cultured in DMEM containing15% FBS, NEAA (1%), basic FGF (bFGF; 5 ng/ml) and Pen/Strep aspreviously described (Ong, et al. (2014). Stem Cell Reports 2, 171-179;Takeda, et al. (2016). Diabetes 65, 1164-1178; Sugii, et al. (2011).Nature Protoc 6, 346-358), and DPSCs were grown in vitro in Poietics™DPSC BulletKit medium (Lonza) according to manufacturer's instructions.Media change for the cells was performed every 2-3 days. All cells werecultured in a humidified incubator at 37° C. in 5% CO₂.

iPS Reprogramming Using Episomal Vectors

Episomal plasmids developed by Yamanaka's lab were obtained fromAddgene: pCXLEhOct3/4-shp53-F (Addgene #27077), pCXLE-hSK (Addgene#27078), pCXLE-hUL (Addgene #27080) and pCXLE-EGFP (Addgene #27082)(Okita, et al. (2011). Nat Methods 8, 409-412). 1×10⁶ cells weresuspended together with 1 μg of each episomal vector in Nucleofectorsolution supplied in the Nucleofector Kit R (Lonza). Then the cells weretransfected with the Program FF-113 on a Nucleofector 2b Device. Thetransfected cells were then cultured in ASC or DPSC medium (MSC medium)supplemented with 0.5 mM sodium butyrate, with daily media change. OnDay 7 dpn, 1×10⁵ viable cells were seeded over MEF feeders (GlobalStem)into one well of a 6-well plate for feeder-based iPS derivation; 2×10⁵viable cells were seeded for feeder-free iPS derivation into one well ofa 6-well plate pre-coated with Matrigel (Corning). The next day, MSCmedium was changed to feeder-based hES medium (DMEM/F12 supplementedwith 20% knock out serum replacement, 1% GlutaMAX, 1% NEAA, Pen/Strep,0.1 mM p-mercaptoethanol and 10 ng/ml b-FGF) or to feeder-free mTeSR1(StemCell Technologies), supplemented with sodium butyrate. At 12 dpn,supplementation of sodium butyrate was stopped, and conditioned furtherwith SMC4 cocktail (consisting of small molecules: PD0325901, CHIR99021,Thiazovivin, and SB431542 (FOCUS Biomolecules)) in hES medium/mTeSR1.This media supplement was continued until initial colony formationbegan.

Fluorescent Probes and Screening

The chemical properties of the BDL library are previously described(Jeong et al., 2015). BDL-E5 is based on4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY), with calculatedmass of 528.3 and absorption maximum/emission maximum of 578/599 nm.Primary, secondary and tertiary screening of fluorescent probes on AiPS,ASCs, DiPS and DPSCs were performed as described previously (Im, et al.(2010). Angew Chem Int Ed Engl 49, 7497-7500; Kang, et al. (2011). NatProtoc 6, 1044-1052) and in the Results section. Unless describedotherwise, BDL-E5 and CDy1 images were acquired by theTetramethylrhodamine (TRITC) channel of ImageXpress Micro High-ContentImaging System, which had the Adaptive Background Correction functionenabled.

Immunofluorescence Live Cell Staining

Reprogrammed cells were immune-stained with fluorescent live cell stainTRA-1-60 (R&D Systems, GloLIVE NL557) as per manufacturer'sinstructions. After incubating with the live staining antibodies for 30min and Hoechst 33342 for 10 min, cells were washed 3 times with PBS andimages with the Cy5 and DAPI channels, respectively, were immediatelycaptured.

Fluorescent Activated Cell Sorting (FACS)

Reprogrammed ASCs and DPSCs, AiPS and DiPS on D7 or D14 dpn were stainedwith BDL-E5 for 1 hour and then harvested using TrypLE and resuspendedin 1×PBS. The cells were then subjected to FACS in the MoFlo XDP CellSorter (Beckman Coulter) under sterile conditions.

RNA Sequencing

At 7 dpn, reprogrammed DPSC2 cells were stained with BDL-E5 andharvested for FACS as mentioned above. 20-30 BDL-E5⁺ and BDL-E5⁻ cellswere collected in 100 μl of 1×PBS; single-cells of DiPS and DPSCs werealso passed through the Cell Sorter and collected for RNA isolation. RNAwas isolated from single cells and cDNA preparation, amplification andquantification were as described in the Supplementary Methods section.Library preparation and sequencing was done by sequencing platform atGenome Institute of Singapore. Paired-end RNA sequencing reads werealigned to the human genome (hg19) using TopHat2-2.0.12 (Kim, et al.(2013). Genome Biol 14, R36) (default parameter). Transcript abundancesat both the gene and isoform levels were estimated by cufflinks-2.2.0(Trapnell, et al. (2010). Nat Biotechnol 28, 511-515) and the expressionwas reported as fragments per kilobase of exon per million fragmentsmapped (FPKM).

Real-Time PCR

Total RNA was extracted using TRizol reagent (Invitrogen) and cDNAconversion was made by the RevertAid H minus first strand cDNA synthesiskit (Fermentas) as per manufacturer's instructions. qPCR was performedusing SYBR Green PCR Master Mix on a StepOnePlus Real-Time PCR System(Applied Biosystems) using the primer pairs shown in Table 1. RelativemRNA was calculated and normalized to the level of GAPDH.

TABLE 1 qPCR primers SEQ SEQ ID ID Gene Forward primer NO:Reverse primer NO: GAPDH CAAGGTCATCCATGACAACTTTG  1 GGCCATCCACAGTCTTCTGG 2 Activin A CTCGGAGATCATCACGTTTG  3 CCTTGGAAATCTCGAAGTGC  4 Lin28GAAGCGCAGATCAAAAGGAG  5 GCTGATGCTCTGGCAGAAGT  6 NanogCCAACATCCTGAACCTCAGC  7 GCTATTCTTCGGCCAGTTG  8 DPPA2TGGTGTCAACAACTCGGTTTG  9 CTCGAACATCGCTGTAATCTGG 10 TGF-ß1GCAGCACGTGGAGCTGTA 11 CAGCCGGTTGCTGAGGTA 12 FN1 CTGGCCGAAAATACATTGTAAA13 CCACAGTCGGGTCAGGAG 14 DNMT3B TACACAGACGTGTCCAACATGGGC 15GGATGCCTTCAGGAATCACACCTC 16 GDF3 AAATGTTTGTGTTGCGGTCA 17TCTGGCACAGGTGTCTTCAG 18 Cdh1 GAAGGTGACAGAGCCTCTGGAT 19GATCGGTTACCGTGATCAAAATC 20 EpCAM1 TGTGTGCGTGGGA 21 TTCAAGATTGGTAAAGCCAGT22 ZEB1 AGCAGTGAAAGAGAAGGGAATGC 23 GGTCCTCTTCAGGTGCCTCAG 24 ZEB2CGCAGCACATGAATCACAGG 25 CGTATCGTTTCGGGATCCGT 26 Snail1TCTGAGGCCAAGGATCTCCA 27 CATTCGGGAGAAGGTCCGAG 28 Snail2TCATCTTTGGGGCGAGTGAG 29 TCCTTGAAGCAACCAGGGTC 30 CREB1ATTGGAAGGAAAGGGGAGGG 31 GGCTTGAACACATCTTGGCA 32 PRKAB2CAGTAGAGTGGGGCAGGAAA 33 TCCCATTTCACATCTGGGCT 34 GATA2AAGGCTCGTTCCTGTTCAGA 35 GGCATTGCACAGGTAGTGG 36 SOX7 GAGCAGTGTGGACACGTACC37 GTCCAGGGGAGACATTTCAG 38 SMA CTGTTCCAGCCATCCTTCAT 39TCATGATGCTGTTGTAGGTGGT 40 AFP AAGAATTTCAGCATGATTTTCCA 41CACCCACTTCATGGTTGCTA 42

CREB1 Overexpression and Silencing by siRNA

CREB1 was overexpressed in ASCs and DPSCs during reprogramming using thecommercially available CREB1 Human cDNA ORF clone (Origene) according tomanufacturer's instructions. Knockdown of CREB1 was achieved using theON-TARGETplus Human CREB1 siRNA—SmartPool (GE Dharmacon) according tomanufacturer's instructions. DPSCs and ASCs were either nucleofectedwith the CREB1 OE along with the episomal reprogramming factors foroverexpression of CREB1 during reprogramming, or transfected withsiCREB1 and then nucleofected with the episomal reprogramming factorsfor silencing of CREB1 during reprogramming.

Generation of Embryoid Bodies and 3-Germ Layer Immunocytochemistry

For spontaneous in vitro differentiation, DPSC-derived iPS (DiPS) cellswere grown to confluency. Using dispase, the cells were resuspended inmedium containing DMEM-F12 supplemented with 10% Knockout SerumReplacement (KOSR), 1% Non-Essential Amino Acid (NEAA) and 1% Glutamax.These cells were transferred to low attachment 6-well plates (GreinerBio One). Media change was made every 3 days. Embryoid bodies (EBs) wereformed as previously described (Kurosawa H (2007). J Biosci Bioeng103(5):389-398), day 8-10 EBs were transferred to a 12-well plateprecoated with 0.1% gelatin and cultured further for 12 more days.Subsequently, the attached EBs were allowed to undergo spontaneousdifferentiation. These differentiated cells were later stained with 3Germ Layer Immunocytochemistry antibodies (Life Technologies) as per themanufacturer's instructions.

Generating and Culturing of Reprogrammed Cells on Cytodex 3Microcarriers

Reprogrammed monolayer ASCs and DPSCs were dissociated into single cellsuspension using Dispase. Single cell suspension was transferred into a6-well Suspension Culture Plate (Greiner bio-one) with Matrigel(Geltrex™, ThermoFisher)-coated Cytodex 3 microcarrier in 4 ml of mTeSR1supplemented with 10 μM of Rock Inhibitor Y27632 (Calbiochem). The platewas placed on an orbital shaker (110 rpm) for at least 2 hours for cellattachment. Afterward, the plate was transferred to static condition andincubated at 37° C./5% CO₂. Media change was carried out with mTeSR1daily thereafter by aspirating 4 ml of spent medium from the well andadding 4 ml of fresh media (80% medium exchange). On Day 7, the cells onmicrocarriers were supplemented and maintained with 4 ml of mTeSR1+SMC4media. 80% medium change was carried out every day.

Fluorescent Probe Staining on Microcarriers

BDL-E5 probe staining of reprogrammed ASCs and DPSCs on microcarrierswas carried out on Day 14 and 21 dpn. The microcarrier culture waswashed twice with sterile D-PBS. Prior to staining, media was changed tomTeSR1 with 500 nM BDL-E5 probe and Alexa Fluor® 488 Mouse anti-humanTRA-1-60 (200× dilution) (BD Pharmingen™). Cultures were incubated for 1hour. The cells were then washed twice with D-PBS and replaced withmTeSR1 prior to imaging. Images were taken using Axio ObserverFluorescent Microscope (Carl Zeiss).

Fluorescent Subcellular Organelle Staining

Reprogrammed DPSCs at 7 dpn on MG were stained for cell organelle markerdyes (Molecular Probes) for endoplasmic reticulum (ER) (ER-Tracker™Green (BODIPY® FL Glibenclamide)), Golgi complex (BODIPY® FL C₅-Ceramide(N-(4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Pentanoyl)Sphingosine)),lysosome (LysoTracker® Green DND-26), or mitochondria (MitoTracker®Green FM). Confocal images were taken to visualize the staining.

iPS Reprogramming Using Viral Methods

iPS cells were also generated from DPSCs with the traditional protocolinvolving retroviral vectors expressing OCT4, SOX2, KLF4, and C-MYC(Sugii, et al. (2011). Nature Protoc 6(3):346-358). BJ human neonatalfibroblasts were reprogrammed using lentiviral OCT4, SOX2, KLF4, andC-MYC as described previously (Toh, et al. (2016). Cell Rep15(12):2597-2607).

Single Cell RNA Isolation for RNA Sequencing

-   -   Prepared Mix A (Lysis), Mix B (RT) and Mix C (PreAmp)

Mix A vol (ul) C1-loading reagent 1 Water 3 Lysis Buffer (0.2% triton-12 X100 in H2O) oligo dT (20 uM) 3.5 rnase inhitor(40 U/ul) 0.5 totalvolume 20

Mix B Vol(ul) Water 1.692 5×-Strand buffer 5.6 DTT (100 mM) 1.4 dNTP 2.8TSO (50 uM) 0.672 rnase inhibitor (40 U/ul) 0.7 RT enzyme (200 U/ul) 2.8MgCl2 (0.5M) 0.336 total volume 16

Mix C Vol(ul) PCR Water 39.5 10× Advantage 2 PCR Buffer 6 50× dNTP Mix2.4 IS PCR Primer(20 uM) 1 50× Advantage 2 Polymerase Mix 2.4 C1 LoadingReagent 2.7 total volume 54

-   -   Dissociated cells into single cell suspension    -   Aliquoted 2 μl mix A into a 200 ul thin PCR tube.    -   Added 1 μl single cell suspension into the PCR tube as        following.

IV ix A 2 μL Prepared Cells 1 μL Subtotal 3 μL

-   -   The lysis step was run by the following program.

Temperature Time 72° C.  3 min  4° C. 10 min 25° C.  1 min  4° C. hold

-   -   Mix B was combined with lysis thermal products from step 4

lysis thermal products 3 μL from above Mix B 4 μL sub total 7 μL

-   -   Following program was run:

Temperature Time 42° C. 90 min 70° C. 10 min  4° C. hold

-   -   Mix C was combined with RT thermal products from step 6

PCR Mix C 4.5 μL 9.0 μL RT Reaction 0.5 μL 1.0 μL subtotal   5 μL  10 μL

-   -   Program was run as follows:

Temperature and Time Cycles 95° C. 1 min 1 95° C. 20 sec 5 58° C. 4 min68° C. 6 min 95° C. 20 sec 9 64° C. 30 sec 68° C. 6 min 95° C. 30 sec 764° C. 30 sec 68° C. 7 min 72° C. 10 min 1  4° C. hold 1

Proliferation Assay

To ensure that BDL-E5 was not toxic to the cells, DPSCs were incubatedwith BDL-E5 (500 nM) for 48 h and 72 h and viable cells were counted ina haemocytometer using the Tryphan Blue method. DPSCs in which CREB1 wasover-expressed or silenced were also counted at 72 h post nucleofectionto determine if gene manipulation has affected the cell proliferation.

Statistical Analysis

All results are presented as means+/−SEM. Statistical analysis wasperformed using t-tests (two sided; paired). Differences with p value<0.05 were considered significant.

Example 1: Screening for a Human Pluripotency-Specific Fluorescent Probe

A high-throughput system using in-house Diversity OrientatedFluorescence Library Approach (DOFLA) was employed to screen 46fluorescent probes that the inventors predicted may specificallyrecognize pluripotent stem cells. To identify fluorescent probes thatdetected human pluripotent stem cells, ASCs, ASC-derived iPS (AiPS)cells, DPSCs, and DPSC-derived iPS (DiPS) cells were used. ASCs andDPSCs were chosen as these cells show relatively high reprogrammingefficiencies and were previously shown to exhibit many of theconventional pluripotent markers, thus serving as stringent negativecontrols for authentic pluripotent stem cells. Cells were seeded onto384-well plates (primary screen) or 96-well plates (secondary andtertiary screen) coated with mouse embryonic fibroblasts (MEF) ormatrigel (MG) for DOFLA screening (FIG. 1A). After 48 h, cells werestained with library probes (500 nM) for 1 h. Fluorescence was imagedusing the ImageXpress System under the following conditions: “No wash”(after 1 h probe incubation), “Wash 0 min” (after one wash with PBS),“Wash 60 min” (after wash and destain for 1 h) and “Wash 180 min” (afterwash and destain for 3 h) (FIG. 1A).

Images were analyzed using the MetaXpress Image Acquisition and Analysissoftware. Following tertiary screening, two probes were shortlisted todevelop further as pluripotent probes: BDL-E5 and CDy1. The chemicalstructure of BDL-E5 is depicted in FIG. 1B. These two probes showedsignificantly increased intensity of fluorescence in human iPS cellscompared with their original somatic cells and MEFs. FIG. 2A showsincreased BDL-E5 staining (No wash) in AiPS colonies grown on MEF- orMG-coated plates, compared with their original somatic cells from theprimary screening. FIG. 8 shows increased CDy1 staining in AiPS cells onMEF- or MG-coated plates compared with ASCs from the primary screen.

Secondary screening was performed to confirm that the probes selectivelystained different iPS colonies. FIG. 2B showed significantly increasedfluorescence intensity of BDL-E5 staining (No wash) in DiPS coloniesgrown on MEF- or MG-coated plates compared with original DPSCs. TheBDL-E5⁺ colonies were also positively stained for the pluripotencymarker TRA-1-60. FIG. 8B showed increased staining of CDy1 and TRA-1-60positive staining in DiPS colonies grown on MEF- or MG-coated platescompared with DPSCs from the secondary screening. Different classes ofprobes were shown to highlight human iPS cells in both feeder andfeeder-free conditions. These results also confirm applicability of CDy1to human cells.

Example 2: BDL-E5 Identified as a Live Fluorescent Probe that DetectsPluripotent Stem Cells

Based on the primary and secondary screenings for live fluorescentprobes that can specifically identify pluripotent cells, BDL-E5 and CDy1were chosen as two probes to further analyze. Tertiary screening wasperformed using the two probes on AiPS colonies under the followingprobe staining conditions: No wash, Wash 0 min, Wash 60 min, and Wash180 min. As shown in FIG. 9A, when AiPS colonies were grown onMEF-coated plates, BDL-E5 staining was greatest with regard to thesignal-to-background ratio under No wash conditions, and CDy1 stainingwas greatest under Wash 60 min or Wash 180 min conditions. When AiPScolonies were grown on MG-coated plates (FIG. 9B), BDL-E5 staining wasgreatest under No wash conditions, and CDy1 staining was greatest underWash 60 min or Wash 180 min conditions. Similar results were alsoobtained with different subject-derived AiPS colonies, as shown in FIG.9C.

Example 3: BDL-E5 can Identify Early Reprogramming Cells

After combining all the results from the primary, secondary, andtertiary screening, the BDL-E5 probe was chosen as the best probeamongst the screened probes, as it did not require washing (thus wasless time- and labor-intensive). Further experiments were carried outusing BDL-E5. To determine whether BDL-E5 identifies the early stages ofpluripotency, ASCs and DPSCs were reprogrammed using nucleofection ofepisomal vectors, and seeded onto MG-coated plates (feeder-free,viral-free reprogramming method). BDL-E5 staining was performed onreprogramming cells at 7, 14, 21 and 28 days post nucleofection (dpn).As shown in FIG. 2C (i) and (ii), the intensity of the fluorescence ofBDL-E5 staining increased with increasing time as iPS colonies wereformed from ASCs or DPSCs. TRA-1-60 staining further confirmed thatcells that stained positive for BDL-E5 were pluripotent. Quantitativedata confirmed this staining as shown in FIG. 2C (iii) and (iv).Interestingly, BDL-E5-positive cells appeared well before colonies werevisible and stained positively for TRA-1-60. BDL-E5-positive cells werefound around 14 dpn (as opposed to 21 dpn for TRA-1-60-positivecolonies) in reprogramming ASCs, while they were observed as early as 7dpn (as opposed to 14 dpn for TRA-1-60) in reprogramming DPSCs.

To confirm that BDL-E5 specifically stains authentic reprogramming cellsthat eventually form colonies, ASCs and DPSCs were reprogrammed usingthe same episomal, feeder-free method. BDL-E5 staining was performed asdescribed previously on the reprogrammed cells every 48 h and imageswere taken from the same field of view daily until iPS colonies formed.FIG. 3 shows representative images at 10, 13, 17, 20, and 24 dpn forreprogrammed ASCs and DPSCs. It is clear that only cells stainingpositive for BDL-E5 formed iPS colonies.

Example 4: BDL-E5⁺ Reprogrammed Cells Generate Higher Quantity andQuality of Ips Cells

Different DPSC cell lines were reprogrammed using the feeder-freeepisomal method, incubated with BDL-E5, and subjected to fluorescenceactivated cell sorting (FACS) at 7 dpn. As shown in FIG. 4A, the bottom10% and top 10% of cells stained with BDL-E5 were sorted, collected, andseeded onto MEF-coated plates. FIG. 10A (i) and (ii) represents theunstained DPSCs.

The cells were allowed to grow for the next two weeks until coloniesappeared. BDL-E5⁺ (top 10%, positively stained) cells gave rise to anincreased number of iPSCs, while BDL-E5⁻ (bottom 10%, negativelystained) cells gave rise to significantly fewer colonies per well (FIG.4B).

Next, the inventors investigated whether the probe was useful inassisting reprogramming selection of obese patient-derived ASCs fromsubcutaneous (SC) and visceral (VS) fat depots. Unlike SC-derived ASCs,VS-derived ASCs exhibit cellular defects, including adipogenesis (Ong etal., 2014; Takeda et al., 2016). It was found that VS-ASCs also showedsubstantial defects in reprogramming, typically resulting in <1 colonybeing formed per well. Interestingly, when ASCs were subjected to FACSwith BDL-E5 at 14 dpn, BDL-E5⁺ and BDL-E5⁻ populations of cells weremore demarcated; SC-ASCs showed higher percentage of cells (˜33%)staining positively for BDL-E5 and ˜11% of cells negatively for BDL-E5(FIG. 4C(i)). VS-ASCs showed a decreased proportion of cells stainingpositively (˜19%) for BDL-E5 and an increased percentage (˜24%) ofnegative cells, as shown in FIG. 4C(ii). FIG. 10A (iii) and (iv)represents the unstained ASCs. BDL-E5⁺ and BDL-E5⁻ cells sorted usingFACS were seeded onto MEF-coated plates. Quantification of the number ofiPS colonies formed after plating clearly indicated that BDL-E5⁺ SC-ASCsgave rise to more colonies than the BDL-E5⁻ population (FIG. 4C(iii) and(iv)). Significantly, at least some BDL-E5⁺ VS-ASCs gave rise to iPScolonies whereas BDL-E5⁻ VS-ASCs did not (FIG. 4C (iii) and (iv)). Thusthese results demonstrate that BDL-E5 staining helps identify the cellpopulation amenable to reprogramming, and increases the chance ofgenerating iPS colonies from difficult-to-reprogram cell types such asVS-ASCs.

To investigate the quality of the BDL-E5⁺ generated iPS cells, the iPScolonies were passaged for several generations. As shown in FIG. 10B(i),ASC-derived BDL-E5⁺ colonies remained well self-renewed and TRA-1-60positive for subsequent passages. However, BDL-E5⁻ cells generated anaverage of only one colony, stained negative with TRA-1-60, and failedto form colonies upon subsequent passages (FIGS. 10B(ii) and C).

Example 5: BDL-E5⁺ Cells have Increased Expression of Pluripotency andEpithelial Markers

The process of cellular reprogramming from somatic to iPS cells involvesmesenchymal-epithelial transition (MET) and increased expression ofpluripotency genes (Li et al., 2010; Samavarchi-Tehrani et al., 2010).DPSCs were reprogrammed and FACS was performed with BDL-E5 at 7 dpn.Expression of pluripotent, epithelial, and mesenchymal genes wasmeasured using qPCR in BDL-E5⁺ (top 10%) and BDL-E5⁻ (bottom 10%) cells.DiPS (dissociated into single cells) and DPSCs (non-reprogrammed)populations were also sorted by FACS and collected as positive andnegative controls, respectively. As shown in FIG. 5A-D, BDL-E5⁺ cellsexhibited increased expression of pluripotency genes, DNMT3B, GDF3,Nanog, LIN28 and DPPA2, and epithelial genes, Cdh1 and EpCAM1, comparedwith BDL-E5⁻ cells. The increased expression of these genes in BDL-E5⁺cells was comparable to that of DiPS cells, and decreased expression ofthese genes in BDL-E5⁻ cells was comparable to that of DPSCs. qPCR wasused to measure expression of mesenchymal genes, ZEB1, ZEB2, Snail1,Snail2, TGF-β1, FN1 and Activin A (FIGS. 5E-H). BDL-E5⁺ cells showeddecreased expression of ZEB2, Snail2 and FN1 compared with BDL-E5⁻cells. BDL-E5⁻ cells showed increased expression of mesenchymal genescomparable to DPSCs. Gene expression measurements using qPCR inreprogrammed ASCs sorted with BDL-E5 at 14 dpn showed increased LIN28and Nanog expression in BDL-E5⁺ cells, while BDL-E5⁻ cells showedsignificantly increased expression of TGF-β1 and slightly increasedexpression of Activin A, with a trend similar to AiPS versus ASCs (FIG.11A).

To confirm that BDL-E5⁺-derived iPS cells are bona fide pluripotentcells, the inventors generated embyoid bodies and allowed the cells tospontaneously differentiate in vitro. The differentiated cells were thensubjected to three-germ layer immunohistochemistry. As shown in FIG.11B(i), the differentiated cells exhibited positive staining for allthree germ layers including ectodermal TUJ1, mesodermal SMA andendodermal AFP. The qPCR analysis also indicated that the spontaneouslydifferentiated cells derived from BDL-E5⁺ iPS cells showed increasedgene expression of ectodermal GATA2, mesodermal SMA and endodermal AFPand SOX7 (FIG. 11B(ii)-(v)). Hence these results supported authenticityof the BDL-E5⁺-derived iPS cells.

Example 6: Bdl-E5 Detects Ips Cells Generated with Common Protocols orThree Dimensional (3d) Culture Conditions, and May Localize to the GolgiComplex

In order to test whether BDL-E5 would be useful for identifyingreprogramming and iPS cells in 3D culture suitable for large scaleproduction, ASCs and DPSCs were reprogrammed and seeded onGeltrex-coated Cytodex 3 microcarriers prior to staining with BDL-E5 andTRA-1-60. As shown in FIG. 11C, BDL-E5 staining was clearly observed inreprogrammed cells in both cell lines, and pluripotency of the iPS cellsformed on the microcarriers was confirmed using TRA-1-60 staining. TheCytodex 3 microcarriers themselves had no background BDL-E5 staining andthe reprogrammed cells were readily distinguishable with intensefluorescence, at both 14 and 21 dpn.

To determine the subcellular organelle localization of BDL-E5 inreprogramming cells, 7 dpn DPSCs on MG were stained for BDL-E5 andorganelle marker dyes for endoplasmic reticulum (ER), Golgi complex,lysosome, or mitochondria. Confocal images showed that BDL-E5 stainingappeared to co-localize significantly with Golgi complex staining, andnot with other organelle markers (FIG. 11D).

We also tested whether BDL-E5 worked for commonly used reprogrammingmethods and cell type. DPSCs were infected with retroviral vectorsexpressing four Yamanaka factors, and plated onto MEF. BDL-E5 similarlystained reprogramming cells as early as 7 days post-infection (dpi),when TRA-1-60 failed to detect any cells (FIG. 12A). In addition, BJhuman fibroblasts were reprogrammed with lentiviral Yamanaka factors.BDL-E5 successfully stained reprogramming, but not non-reprogrammingcells, and staining was stronger than that of TRA-1-60 (FIGS. 12B andC).

Example 7: RNA-Sequencing Analysis Reveals Early Reprogramming Markersin BDL-E5+ Cells

To identify classes of novel genes that might be involved in earlyreprogramming stages defined by BDL-E5, RNA sequencing was performed onBDL-E5⁺ and BDL-E5⁻ DPSCs sorted at 7 dpn, using DPSCs and DiPS cells asreference controls. Genes showing statistical significance (p<0.05)and >2 fold change were selected for analysis and, overall, 386 genes(shown in Table 2) were significantly differentially expressed (106upregulated and 280 downregulated) in BDL-E5⁺ versus BDL-E5⁻ sortedcells as shown in a heatmap and Venn diagram (FIGS. 6A and 6B). Furtheranalysis of the 386 differentially expressed genes was carried out.Among the BDL-E5+ upregulated genes, 31 genes were expressed higher and57 genes were expressed lower in DiPS cells compared with DPSCs. On theother hand, 117 genes were expressed higher and 139 genes were expressedlower in DiPS cells than DPSCs among the BDL-E5⁺ downregulated genes.Annotation using Ingenuity Pathway indicated that differentiallyregulated genes in BDL-E5⁺ versus BDL-E5⁻ cells were associated with“ebmryonic development”, “organismal development,” and “tissuedevelopment” categories. Top canonical pathway and molecular cellularfunctions included “BMP signaling pathway,” “FGF signaling,”“cell-to-cell signaling and interaction,” “cellular assembly andorganization,” and “cellular growth and proliferation” (FIG. 13A).Another analysis was performed using Metascape and demonstrated that thetop enriched clusters between BDL-E5⁺ and BDL-E5⁻ cells included“lamellipodium morphogenesis,” “positive regulation of organelleorganization,” “regulation of transporter activity,” “cell morphogenesisinvolved in neuron differentiation,” and “embryo development” (FIG.13B).

Among these, the inventors were particularly interested in CREB1 andPRKAB2 genes due to their potential involvement in the metabolicreprogramming process (FIGS. 6A and 6C).

Expression of CREB1 was significantly upregulated in BDL-E5⁺ cellscompared with BDL-E5⁻ cells. CREB1 was also upregulated in DiPS cells.PRKAB2 was downregulated in both DiPS and BDL-E5⁺ cells. Gene expressionwas further confirmed by qPCR as shown in FIG. 6D.

Based on these results, the inventors hypothesized that the pathwayregulated by CREB1 may be involved in the early reprogramming process ofcells that are marked by BDL-E5.

TABLE 2 List of the 386 genes differentially expressed in DiPS, DPSC,BDL-E5⁺, BDL-E5⁻ Gene ID Gene Symbol DiPS DPSC BDL-E5⁺ BDL-E5⁻ p valueENSG00000029153.10 ARNTL2 0.00 0.04 0.37 4.82 0.0188 ENSG00000030304.8MUSK 0.00 0.54 0.03 9.01 0.0089 ENSG00000059378.8 PARP12 0.00 19.17 9.5586.06 0.0148 ENSG00000068724.11 TTC7A 0.00 15.83 1.33 40.91 0.04115ENSG00000069869.11 NEDD4 0.00 5.41 5.13 42.48 0.00865 ENSG00000076258.5FMO4 0.00 9.28 0.00 1.25 0.00815 ENSG00000090975.8 PITPNM2 0.00 8.280.19 6.26 0.02695 ENSG00000102078.11 SLC25A14 0.00 54.00 1.34 146.610.001 ENSG00000109667.7 SLC2A9 0.00 0.00 1.26 0.00 5.00E−05ENSG00000117152.9 RGS4 0.00 0.00 0.45 0.00 0.0056 ENSG00000119725.13ZNF410 0.00 0.26 30.07 0.61 0.0031 ENSG00000123243.10 ITIH5 0.00 1.290.00 0.64 0.00055 ENSG00000130382.7 MLLT1 0.00 16.41 0.00 104.615.00E−05 ENSG00000134007.3 ADAM20 0.00 0.00 5.27 0.00 0.02535ENSG00000134539.12 KLRD1 0.00 0.00 0.54 0.00 0.0143 ENSG00000135362.9PRR5L 0.00 5.56 0.80 21.84 0.00695 ENSG00000138772.8 ANXA3 0.00 0.003.12 128.24 0.0193 ENSG00000141255.8 SPATA22 0.00 0.00 0.00 0.69 0.0098ENSG00000143127.8 ITGA10 0.00 2.80 0.58 0.00 0.01655 ENSG00000148444.11COMMD3 0.00 63.25 35.07 273.40 0.03705 ENSG00000152128.13 TMEM163 0.000.00 0.00 68.59 5.00E−05 ENSG00000154678.12 PDE1C 0.00 6.75 35.24 6.840.0365 ENSG00000158806.9 NPM2 0.00 0.00 0.00 32.06 6.00E−04ENSG00000159314.7 ARHGAP27 0.00 0.00 0.00 2.70 0.001 ENSG00000161681.11SHANK1 0.00 0.00 0.00 0.55 0.00055 ENSG00000164463.8 CREBRF 0.00 7.4519.68 0.95 0.0135 ENSG00000167136.6 ENDOG 0.00 3.97 2.83 0.00 0.03695ENSG00000167925.11 GHDC 0.00 3.88 0.03 50.77 3.00E−04 ENSG00000168405.10CMAHP 0.00 0.01 0.00 211.45 5.00E−05 ENSG00000172733.10 PURG 0.00 0.190.00 4.09 0.04485 ENSG00000173083.10 HPSE 0.00 0.00 0.43 30.85 0.048ENSG00000178685.9 PARP10 0.00 2.15 0.97 214.51 0.03005 ENSG00000183644.9C11orf88 0.00 2.26 0.67 0.00 0.01075 ENSG00000184588.13 PDE4B 0.00 37.0210.35 158.90 0.0108 ENSG00000186998.11 EMID1 0.00 0.00 0.00 0.40 0.0085ENSG00000196843.11 ARID5A 0.00 46.00 4.66 131.21 0.0251ENSG00000197182.8 FLJ27365 0.00 3.53 0.49 23.02 0.036 ENSG00000197584.7KCNMB2 0.00 0.00 0.00 0.59 0.02015 ENSG00000198520.6 C1orf228 0.00 0.000.00 79.96 0.04935 ENSG00000205746.5 RP11- 0.00 0.00 0.00 2.11 0.03321212A22.1 ENSG00000206127.6 GOLGA8O 0.00 0.92 1.75 0.00 0.04085ENSG00000215527.3 AP005482.1 0.00 0.00 0.00 333.91 0.0039ENSG00000218996.1 RP1-99E18.2 0.00 0.00 0.00 4.86 0.04195ENSG00000224080.1 UBE2FP1 0.00 0.58 0.00 1.16 0.017 ENSG00000224623.1RP11-247I13.8 0.00 0.00 2.28 0.00 0.02075 ENSG00000225383.2 SFTA1P 0.000.00 0.00 11.76 0.0279 ENSG00000225920.2 RIMKLBP2 0.00 0.00 2.14 0.000.01615 ENSG00000227953.2 RP11-439E19.3 0.00 3.29 1.70 0.00 0.0181ENSG00000229052.2 RP11-386I23.1 0.00 0.66 0.91 0.00 0.02955ENSG00000229124.2 VIM-AS1 0.00 0.02 0.53 0.00 0.01325 ENSG00000229325.1ACAP2-IT1 0.00 0.00 14.51 0.00 0.03095 ENSG00000229692.3 SOS1-IT1 0.000.00 0.00 10.49 0.0421 ENSG00000229808.1 RP11-456P18.2 0.00 0.00 0.910.00 0.0258 ENSG00000230001.1 RP11-70J12.1 0.00 2.82 12.54 0.00 0.03395ENSG00000232116.2 RP11-187C18.2 0.00 15.03 0.00 11.70 0.04865ENSG00000234636.1 MED14-AS1 0.00 0.00 0.00 1.40 0.0281 ENSG00000237654.1AP003025.2 0.00 1.14 0.00 22.22 0.04965 ENSG00000237803.1 LINC00211 0.000.00 0.00 0.29 0.03995 ENSG00000238113.2 RP11-262H14.1 0.00 0.28 0.003.42 0.0337 ENSG00000240695.1 RP11-102M11.1 0.00 20.15 0.00 1.77 0.03325ENSG00000241295.1 ZBTB20-AS2 0.00 12.76 23.50 0.00 0.0494ENSG00000242154.1 RP4-778K6.3 0.00 0.00 12.86 0.00 0.045ENSG00000243251.4 PGBD3 0.00 9.15 13.42 0.00 0.0263 ENSG00000243305.1RP11-362A9.3 0.00 1.72 0.00 4.01 0.01435 ENSG00000248664.1 CTC-498J12.30.00 0.00 0.00 0.40 0.0166 ENSG00000249593.2 CTB-46B19.2 0.00 0.00 0.320.00 0.0388 ENSG00000251381.2 LINC00958 0.00 0.00 0.28 0.00 0.0079ENSG00000255139.1 AP000442.1 0.00 0.00 0.00 178.60 0.0076ENSG00000256025.1 CACNA1C-AS4 0.00 4.99 0.00 41.02 0.0065ENSG00000256390.1 AC092143.1 0.00 0.00 0.41 0.00 0.01655ENSG00000256469.1 RP11-856F16.2 0.00 1.63 0.00 6.09 0.0419ENSG00000258978.1 HIF1AP1 0.00 0.00 0.00 124.37 0.01085ENSG00000259948.2 RP11-326A19.5 0.00 0.00 0.68 0.00 0.02955ENSG00000260946.1 RP11-407G23.3 0.00 27.94 0.00 74.33 0.0244ENSG00000261355.1 RP11-698N11.4 0.00 0.17 2.90 0.00 5.00E−05ENSG00000261777.1 RP11-529K1.2 0.00 0.00 0.70 0.00 0.0238ENSG00000262211.1 CTD-2031P19.5 0.00 0.00 4.09 0.00 0.0189ENSG00000267395.1 AC074212.6 0.00 7.25 2.62 0.00 0.02365ENSG00000267515.1 RP11-861E21.3 0.00 0.00 27.81 0.00 0.0282ENSG00000267811.1 RP11- 0.00 1.03 6.34 0.00 0.011 727F15.11ENSG00000269997.1 RP11-214K3.21 0.00 0.00 0.00 211.50 0.0488ENSG00000272533.1 SNORA28 0.00 0.00 519.97 0.00 0.0036 ENSG00000272991.1AF129408.17 0.00 31.84 31.93 0.00 0.0139 ENSG00000273297.1 RP11-38M8.10.00 0.76 7.39 0.00 0.0271 ENSG00000273384.1 RP5-1098D14.1 0.00 0.000.00 138.30 0.01545 ENSG00000271741.1 ZMYM6 0.00 0.03 1.77 0.00 0.04815ENSG00000250802.2 ZBED3-AS1 0.00 0.00 1.41 0.00 0.0187ENSG00000172716.12 SLFN11 0.00 4.19 23.14 0.40 0.0229 ENSG00000196724.8ZNF418 0.00 0.98 0.00 0.77 0.01825 ENSG00000107738.15 C10orf54 0.0049.53 55.13 11.04 0.0382 ENSG00000155066.11 PROM2 0.00 0.00 0.00 0.420.00015 ENSG00000144810.11 COL8A1 0.01 24.25 27.76 110.32 0.03055ENSG00000163412.8 EIF4E3 0.01 1.09 1.39 0.07 0.01515 ENSG00000113296.10THBS4 0.01 0.00 0.00 12.35 0.0023 ENSG00000123552.13 USP45 0.01 2.6417.58 1.72 0.01925 ENSG00000137809.12 ITGA11 0.01 54.05 43.73 178.380.03325 ENSG00000175787.12 ZNF169 0.02 0.01 0.00 2.61 0.0192ENSG00000105605.3 CACNG7 0.02 0.05 0.00 0.43 0.0316 ENSG00000198690.5FAN1 0.02 0.49 2.89 0.25 0.03 ENSG00000142794.14 NBPF3 0.02 4.98 0.050.37 0.01015 ENSG00000006283.13 CACNA1G 0.03 0.04 0.00 0.58 0.0013ENSG00000132256.14 TRIM5 0.03 37.19 1.75 38.68 0.0078 ENSG00000213073.4RP11-288H12.3 0.03 0.00 0.00 36.39 0.00625 ENSG00000214176.5 PLEKHM1P0.04 5.89 0.01 4.55 0.0222 ENSG00000135297.11 MTO1 0.04 0.16 0.87 34.480.0013 ENSG00000259571.1 BLID 0.04 104.78 2.91 0.00 0.03145ENSG00000110756.13 HPS5 0.05 0.26 33.82 6.38 0.04325 ENSG00000239713.3APOBEC3G 0.05 0.13 3.51 0.04 0.03025 ENSG00000159403.11 C1R 0.06 1182.24205.29 848.86 0.0167 ENSG00000112769.14 LAMA4 0.07 121.77 38.18 340.150.00175 ENSG00000142330.15 CAPN10 0.07 30.00 0.78 165.69 5.00E−05ENSG00000256594.3 RP11-705C15.2 0.07 0.66 0.00 4.84 0.0411ENSG00000138639.13 ARHGAP24 0.07 0.00 85.90 0.34 0.00665ENSG00000181938.9 GINS3 0.08 0.07 0.00 0.64 0.0057 ENSG00000219470.1RP3-337H4.6 0.08 0.17 0.83 0.00 0.0459 ENSG00000272899.1 RP11-309L24.90.08 0.15 9.22 0.00 0.00605 ENSG00000134802.13 SLC43A3 0.09 0.14 1.480.06 0.0071 ENSG00000204991.6 SPIRE2 0.09 3.73 0.00 0.37 0.00115ENSG00000261552.1 RP11-264B17.5 0.09 27.02 0.00 64.44 0.0378ENSG00000235865.2 GSN-AS1 0.10 5.55 0.08 9.57 0.04845 ENSG00000183023.14SLC8A1 0.11 19.12 3.02 65.61 0.0257 ENSG00000146373.12 RNF217 0.11 8.191.31 6.25 0.02805 ENSG00000121964.10 GTDC1 0.11 4.26 2.52 76.83 0.0119ENSG00000203836.7 NBPF24 0.13 0.04 0.00 2.79 0.02085 ENSG00000175984.10DENND2C 0.15 0.01 0.48 9.77 0.0095 ENSG00000119681.7 LTBP2 0.16 8.835.10 44.40 0.01055 ENSG00000269242.1 CTD- 0.18 7.03 35.91 0.00 0.023552192J16.22 ENSG00000183655.11 KLHL25 0.18 0.14 0.00 1.15 0.0423ENSG00000196159.7 FAT4 0.18 8.16 4.10 16.21 0.03155 ENSG00000167972.9ABCA3 0.20 0.00 0.00 2.58 0.0303 ENSG00000140471.12 LINS 0.21 78.54 0.5082.48 0.01345 ENSG00000235034.2 C19orf81 0.22 2.90 0.00 0.74 0.0489ENSG00000138134.7 STAMBPL1 0.22 0.01 0.00 7.50 0.03875ENSG00000151883.12 PARP8 0.23 0.00 0.01 0.26 0.00025 ENSG00000183405.5RPS7P1 0.23 0.21 0.54 0.00 0.03545 ENSG00000213707.2 HMGB1P10 0.23 0.480.00 5.47 0.01645 ENSG00000204084.8 INPP5B 0.23 0.54 0.65 17.81 0.03345ENSG00000160469.12 BRSK1 0.24 0.00 0.00 1.25 0.0152 ENSG00000072518.16MARK2 0.26 0.18 1.14 24.73 0.02035 ENSG00000146263.7 MMS22L 0.27 0.250.00 5.45 5.00E−05 ENSG00000021645.13 NRXN3 0.30 0.02 8.17 0.00 5.00E−05ENSG00000185278.10 ZBTB37 0.30 2.64 0.01 0.59 0.02725 ENSG00000139926.11FRMD6 0.35 26.83 55.29 458.21 0.00925 ENSG00000223953.3 C1QTNF5 0.36204.94 201.46 1600.38 0.00135 ENSG00000166311.5 SMPD1 0.38 94.15 112.34585.07 0.02725 ENSG00000129657.10 SEC14L1 0.40 19.75 73.50 19.64 0.03895ENSG00000105483.12 CARD8 0.45 22.43 0.66 14.91 0.02 ENSG00000161958.6FGF11 0.51 1.71 0.33 18.23 0.0171 ENSG00000167615.12 LENG8 0.54 24.861.75 177.96 0.01025 ENSG00000160828.13 STAG3L2 0.58 9.10 0.27 5.500.0252 ENSG00000260793.2 RP5-882C2.2 0.63 2.04 3.70 0.00 0.01455ENSG00000106648.9 GALNTL5 0.64 0.06 0.00 0.67 0.0041 ENSG00000157869.10RAB28 0.64 1.06 14.07 73.16 0.0405 ENSG00000158773.10 USF1 0.69 13.1413.84 0.00 0.022 ENSG00000229809.4 ZNF688 0.72 5.79 0.76 40.82 0.013ENSG00000232931.1 LINC00342 0.73 6.33 8.56 64.96 0.02945ENSG00000141576.10 RNF157 0.84 0.00 0.5 0.01 0.04825 ENSG00000255248.2RP11-166D19.1 0.86 138.45 33.59 207.41 0.0227 ENSG00000118762.3 PKD20.86 28.76 39.41 8.30 0.0392 ENSG00000147457.9 CHMP7 0.88 11.38 37.024.23 0.0461 ENSG00000106608.12 URGCP 0.89 1.17 51.04 0.35 0.0022ENSG00000162928.8 PEX13 0.95 4.92 4.36 146.84 0.0077 ENSG00000090674.11MCOLN1 1.05 60.52 0.76 19.86 0.0332 ENSG00000073711.6 PPP2R3A 1.11 6.574.59 29.75 0.04325 ENSG00000229153.1 EPHA1-AS1 1.12 0.24 5.52 0.000.00925 ENSG00000232586.1 RP11-46A10.4 1.16 0.06 0.00 0.58 0.01455ENSG00000122481.12 RWDD3 1.21 63.16 39.82 225.43 0.0445ENSG00000136720.6 HS6ST1 1.21 60.37 23.29 123.76 0.0454ENSG00000188130.9 MAPK12 1.29 51.40 65.20 1.23 0.0202 ENSG00000133250.9ZNF414 1.31 0.32 0.00 24.34 0.0045 ENSG00000160352.11 ZNF714 1.32 0.240.00 0.97 0.0136 ENSG00000172725.9 CORO1B 1.34 1.67 19.94 1.33 0.0099ENSG00000236526.1 RP4-742J24.2 1.35 12.53 1.22 0.00 0.0318ENSG00000150712.6 MTMR12 1.35 5.44 6.96 0.39 0.02425 ENSG00000074527.7NTN4 1.36 22.30 203.79 47.63 0.03745 ENSG00000197016.7 ZNF470 1.52 0.080.13 4.08 0.0473 ENSG00000164338.5 UTP15 1.56 2.37 1.61 57.46 0.0041ENSG00000206560.6 ANKRD28 1.66 65.12 5.10 79.01 0.006 ENSG00000075539.9FRYL 1.85 170.14 5.05 44.67 0.02035 ENSG00000268093.1 AC022154.7 1.881.09 0.00 0.50 0.0292 ENSG00000125962.10 ARMCX5 1.92 0.11 1.52 59.320.03555 ENSG00000129911.4 KLF16 1.93 3.55 0.99 32.26 0.04305ENSG00000164877.14 MICALL2 1.96 62.11 32.67 229.73 0.0311ENSG00000257647.1 RP11-701H24.3 2.00 0.94 0.00 10.99 0.02645ENSG00000115966.12 ATF2 2.09 7.12 41.34 5.69 0.0267 ENSG00000160216.14AGPAT3 2.12 4.83 5.54 47.29 0.02025 ENSG00000256525.2 POLG2 2.16 10.310.43 15.15 0.0276 ENSG00000149929.11 HIRIP3 2.26 4.78 25.38 0.03 0.0474ENSG00000174684.6 B3GNT1 2.30 39.47 17.05 105.05 0.0474ENSG00000105559.7 PLEKHA4 2.40 28.99 8.83 83.57 0.02915ENSG00000187609.11 EXD3 2.49 30.13 4.28 118.89 0.00825ENSG00000185046.14 ANKS1B 2.49 0.51 0.44 0.00 0.0165 ENSG00000184381.14PLA2G6 2.51 0.19 0.00 1.43 0.00255 ENSG00000136436.10 CALCOCO2 2.6622.13 46.79 333.30 0.0178 ENSG00000213918.6 DNASE1 2.66 8.69 16.15 1.030.04625 ENSG00000161395.8 PGAP3 2.77 4.93 0.68 54.65 0.03815ENSG00000204196.4 AC011737.2 2.78 2.52 0.75 99.18 0.02925ENSG00000103111.10 MON1B 2.83 22.08 44.23 1.76 0.0113 ENSG00000269279.1AL136376.1 2.91 4.08 0.00 5.41 0.01205 ENSG00000139579.8 NABP2 2.94 6.260.77 47.39 0.03925 ENSG00000154370.9 TRIM11 3.17 9.59 0.73 30.51 0.0225ENSG00000104081.9 BMF 3.20 0.45 0.03 47.46 3.00E−04 ENSG00000145012.8LPP 3.56 10.43 10.98 120.27 0.0096 ENSG00000113716.8 HMGXB3 3.67 1.670.70 31.38 0.02915 ENSG00000241258.2 CRCP 3.73 38.44 21.51 2.48 0.0467ENSG00000198380.8 GFPT1 3.79 4.53 9.62 93.28 0.0223 ENSG00000166912.12MTMR10 3.90 8.82 5.29 33.69 0.0355 ENSG00000267542.1 RP11-697E22.1 3.9511.63 12.97 0.00 0.0377 ENSG00000253251.2 CTC-534A2.2 4.15 5.41 0.003.86 0.04345 ENSG00000138035.10 PNPT1 4.37 0.72 0.87 19.99 0.0243ENSG00000105321.8 CCDC9 4.37 0.02 1.11 0.00 0.007 ENSG00000103260.4METRN 4.43 48.99 2.57 133.57 0.008 ENSG00000269388.1 AC018755.16 4.480.00 0.00 1.31 0.0316 ENSG00000198954.4 KIAA1279 4.61 19.18 8.65 70.080.0416 ENSG00000075420.8 FNDC3B 4.80 20.14 40.04 230.59 0.0327ENSG00000157353.12 FUK 4.81 5.11 0.47 34.19 0.0492 ENSG00000256904.1A2ML1-AS2 4.91 0.00 0.00 1.29 0.01695 ENSG00000177873.8 ZNF619 5.02 0.020.00 1.40 0.0155 ENSG00000125846.11 ZNF133 5.12 2.29 0.71 34.67 0.00535ENSG00000118518.11 RNF146 5.16 7.44 10.78 121.79 0.01285ENSG00000160055.15 TMEM234 5.22 0.97 6.59 0.19 0.04965 ENSG00000132004.8FBXW9 5.31 21.06 2.55 67.37 0.03545 ENSG00000131724.6 IL13RA1 5.32 9.0921.38 4.19 0.0234 ENSG00000196810.4 CTBP1-AS2 5.33 14.28 0.12 82.990.00365 ENSG00000186862.13 PDZD7 5.49 1.83 0.15 4.38 0.04035ENSG00000243335.4 KCTD7 5.51 0.13 0.36 13.44 0.0414 ENSG00000078246.11TULP3 5.68 4.64 5.79 194.84 0.02755 ENSG00000198055.6 GRK6 5.69 1.720.07 5.69 0.03345 ENSG00000149930.13 TAOK2 6.12 3.42 0.55 14.52 0.0332ENSG00000140367.7 UBE2Q2 6.24 16.84 3.26 21.11 0.04405ENSG00000175866.11 BAIAP2 6.27 3.32 57.96 4.18 0.00785 ENSG00000167257.6RNF214 6.44 4.30 1.46 15.98 0.02625 ENSG00000175567.4 UCP2 6.48 0.000.00 1.83 0.02095 ENSG00000033178.8 UBA6 6.52 24.99 7.56 61.16 0.01225ENSG00000157456.3 CCNB2 6.99 0.00 0.00 1.33 0.04925 ENSG00000145860.7RNF145 7.09 17.06 7.28 92.04 0.03475 ENSG00000146243.9 IRAK1BP1 7.161.61 6.18 0.07 0.03085 ENSG00000130669.13 PAK4 7.20 2.21 13.55 0.930.02925 ENSG00000180881.15 CAPS2 7.38 0.45 0.10 18.70 0.00205ENSG00000079739.11 PGM1 7.57 15.02 30.84 179.18 0.03445ENSG00000108830.7 RND2 7.63 0.00 0.00 0.58 0.04305 ENSG00000168528.7SERINC2 8.13 12.66 23.44 193.70 0.03645 ENSG00000131196.13 NFATC1 8.172.59 0.93 47.76 8.00E−04 ENSG00000131791.6 PRKAB2 8.24 10.26 10.18 69.190.0279 ENSG00000176105.9 YES1 8.75 7.29 5.68 33.89 0.0442ENSG00000100479.8 POLE2 8.90 5.62 11.80 0.00 0.0459 ENSG00000124782.15RREB1 8.96 25.10 1.97 89.52 0.02475 ENSG00000151746.9 BICD1 9.06 1.760.61 11.99 0.02745 ENSG00000110422.7 HIPK3 9.20 10.84 5.34 24.44 0.049ENSG00000168758.6 SEMA4C 9.32 0.83 0.40 0.00 0.01885 ENSG00000107779.7BMPR1A 9.82 2.90 1.69 13.06 0.0123 ENSG00000117713.13 ARID1A 9.88 5.752.74 27.83 0.0214 ENSG00000151348.9 EXT2 9.93 57.52 38.28 9.35 0.03815ENSG00000001631.10 KRIT1 10.04 7.02 5.06 134.21 0.0125 ENSG00000177885.9GRB2 10.04 88.12 31.28 338.39 0.03615 ENSG00000131323.10 TRAF3 10.263.50 3.35 0.07 0.028 ENSG00000110075.10 PPP6R3 10.72 23.86 16.20 87.310.024 ENSG00000163935.9 SFMBT1 11.68 2.11 0.21 89.49 0.01245ENSG00000006468.9 ETV1 11.78 0.00 0.00 8.05 5.00E−05 ENSG00000184347.10SLIT3 11.81 12.31 16.97 171.65 0.04765 ENSG00000119689.10 DLST 11.921.88 30.21 0.07 0.00345 ENSG00000181027.6 FKRP 12.13 4.73 1.48 106.760.04255 ENSG00000113312.6 TTC1 12.40 126.88 354.60 72.10 0.023ENSG00000177943.9 MAMDC4 12.55 7.26 0.00 1.00 0.0257 ENSG00000105127.4AKAP8 12.66 15.52 1.54 38.75 0.02535 ENSG00000125459.10 MSTO1 12.88 1.590.19 5.84 0.0181 ENSG00000161202.13 DVL3 12.95 18.30 2.19 51.52 0.01915ENSG00000121152.5 NCAPH 14.46 0.00 0.00 63.91 0.01595 ENSG00000156970.8BUB1B 14.58 0.00 0.06 2.81 0.047 ENSG00000138190.12 EXOC6 14.73 0.090.10 10.43 0.0127 ENSG00000153250.13 RBMS1 15.14 144.93 48.51 235.580.01575 ENSG00000124155.12 PIGT 15.15 109.11 95.81 353.88 0.04185ENSG00000196526.6 AFAP1 15.19 26.76 5.47 92.03 0.0186 ENSG00000170310.10STX8 16.50 40.57 267.05 1088.17 0.0152 ENSG00000145014.13 TMEM44 18.5828.86 14.34 0.73 0.0469 ENSG00000088833.13 NSFL1C 18.81 16.25 87.23 9.000.042 ENSG00000127580.11 WDR24 19.13 6.16 0.35 36.69 0.04805ENSG00000196313.7 POM121 19.33 2.06 0.11 39.20 0.0261 ENSG00000103168.12TAF1C 19.36 21.51 0.89 26.80 0.0283 ENSG00000135506.11 OS9 19.36 68.8529.56 242.73 0.02475 ENSG00000113734.13 BNIP1 19.53 2.07 21.33 0.670.04895 ENSG00000164543.5 STK17A 19.79 2.70 3.01 74.86 0.0051ENSG00000163006.7 CCDC138 20.21 0.19 0.25 0.00 0.0038 ENSG00000151657.7KIN 20.34 66.80 11.57 124.07 0.0316 ENSG00000126787.8 DLGAP5 20.49 0.000.00 0.62 0.01385 ENSG00000137942.12 FNBP1L 20.80 14.61 6.42 36.330.04555 ENSG00000158195.6 WASF2 20.87 99.75 79.55 679.26 0.03235ENSG00000243725.2 TTC4 21.15 4.71 9.86 78.68 0.02515 ENSG00000168916.11ZNF608 21.36 5.43 0.00 0.85 0.00165 ENSG00000133812.10 SBF2 21.42 45.3439.90 6.14 0.0488 ENSG00000106012.13 IQCE 21.84 55.60 3.61 47.81 0.0439ENSG00000139971.11 C14orf37 22.26 9.61 1.15 50.49 0.0086ENSG00000138867.12 GUCD1 22.36 48.68 10.17 224.51 0.04415ENSG00000267228.2 IER3IP1 22.96 32.14 7.42 0.00 0.01955ENSG00000176390.10 CRLF3 23.22 3.67 0.06 6.89 0.00585 ENSG00000101290.9CDS2 23.78 0.94 51.41 3.45 0.03045 ENSG00000165030.3 NFIL3 23.84 32.6630.14 219.23 0.0146 ENSG00000198363.11 ASPH 23.84 230.74 326.46 97.560.019 ENSG00000062650.13 WAPAL 24.14 56.28 9.24 121.79 0.02625ENSG00000172273.8 HINFP 24.42 16.38 0.06 0.26 0.04175 ENSG00000224019.1RPL21P32 24.50 0.00 0.00 173.08 0.0054 ENSG00000145194.13 ECE2 24.6333.05 0.18 16.46 0.04065 ENSG00000141985.5 SH3GL1 24.78 49.28 15.28145.87 0.01315 ENSG00000157895.7 C12orf43 25.94 44.58 1.25 15.65 0.00735ENSG00000138663.4 COPS4 26.31 22.05 31.53 202.21 0.0262ENSG00000169504.10 CLIC4 26.70 140.30 205.12 48.45 0.01695ENSG00000132676.11 DAP3 27.14 114.80 48.07 317.13 0.01545ENSG00000103275.14 UBE21 28.48 34.42 17.49 91.88 0.0274ENSG00000122482.16 ZNF644 28.54 18.23 27.17 3.57 0.04385ENSG00000127946.12 HIP1 29.77 5.83 3.13 75.14 0.0379 ENSG00000118260.10CREB1 31.96 4.70 42.52 3.49 0.0027 ENSG00000152413.10 HOMER1 32.53 4.811.83 90.78 0.00105 ENSG00000090686.11 USP48 33.75 118.84 39.13 133.690.0307 ENSG00000075131.5 TIPIN 33.93 1.21 0.17 3.45 0.0392ENSG00000124574.10 ABCC10 34.82 0.80 5.09 0.00 0.02555ENSG00000136153.15 LMO7 35.40 384.16 247.03 1215.23 0.0112ENSG00000034677.7 RNF19A 37.93 22.98 13.14 163.02 0.03895ENSG00000063244.8 U2AF2 38.77 3.04 15.08 1.20 0.043 ENSG00000152942.14RAD17 39.05 6.99 5.55 158.97 0.0465 ENSG00000113658.12 SMAD5 39.06 60.6218.06 251.89 0.04935 ENSG00000066923.13 STAG3 41.21 1.55 0.00 2.210.0047 ENSG00000010318.15 PHF7 44.31 5.43 0.00 3.88 0.012ENSG00000157500.6 APPL1 44.31 62.47 47.78 4.28 0.01785ENSG00000144524.13 COPS7B 45.83 34.73 7.04 73.72 0.00975ENSG00000116679.11 IVNS1ABP 46.20 33.51 14.92 125.37 0.01845ENSG00000134363.7 FST 46.86 248.92 116.59 481.74 0.0361ENSG00000168724.10 DNAJC21 47.19 158.17 33.90 356.35 0.0107ENSG00000081320.6 STK17B 47.87 21.00 65.30 6.08 0.02835ENSG00000077782.15 FGFR1 53.88 115.95 47.08 226.61 0.019ENSG00000131037.10 EPS8L1 54.02 0.00 0.00 68.85 5.00E−05ENSG00000100796.13 SMEK1 54.56 29.85 9.80 113.19 0.007 ENSG00000112576.8CCND3 56.84 12.85 41.94 216.48 0.04555 ENSG00000077312.4 SNRPA 57.785.97 3.16 100.41 0.0107 ENSG00000038382.13 TRIO 58.79 62.20 10.88 116.430.0124 ENSG00000117616.13 C1orf63 59.63 357.82 133.16 732.99 0.0166ENSG00000112531.12 QKI 61.47 6.84 5.88 47.98 0.0346 ENSG00000185864.12NPIPB4 61.65 24.39 3.66 36.08 0.0051 ENSG00000140632.12 GLYR1 62.4125.16 3.82 127.73 9.00E−04 ENSG00000064607.12 SUGP2 66.35 40.94 18.892.14 0.03335 ENSG00000163697.12 APBB2 68.17 9.71 44.92 5.15 0.0026ENSG00000100836.6 PABPN1 68.71 136.49 36.31 186.46 0.0399ENSG00000214021.11 TTLL3 70.98 33.52 2.43 439.85 0.0016ENSG00000103091.10 WDR59 76.24 8.34 2.46 55.56 0.04075 ENSG00000242114.1MTFP1 76.61 0.00 1.52 147.89 0.0217 ENSG00000116809.7 ZBTB17 79.34 10.401.01 30.96 0.0256 ENSG00000054654.11 SYNE2 81.62 2.70 0.50 3.68 0.0446ENSG00000145016.9 KIAA0226 89.15 11.86 9.78 139.03 0.0266ENSG00000109920.8 FNBP4 92.79 42.91 33.64 230.39 0.0158ENSG00000119048.3 UBE2B 103.42 39.37 100.96 682.64 0.0443ENSG00000172889.11 EGFL7 106.66 0.63 1.09 230.19 0.01655ENSG00000101115.8 SALL4 106.90 5.55 0.00 74.06 5.00E−05ENSG00000089006.12 SNX5 107.25 102.70 44.24 405.60 0.01565ENSG00000223745.3 RP4-717I23.3 108.07 11.50 55.98 5.28 0.029ENSG00000078808.12 SDF4 111.39 113.08 19.25 419.99 0.0034ENSG00000150753.7 CCT5 114.70 49.14 79.81 294.12 0.0456ENSG00000074054.13 CLASP1 116.42 4.10 21.39 548.20 0.0067ENSG00000176022.3 B3GALT6 121.05 36.08 29.74 238.61 0.0345ENSG00000118058.16 KMT2A 131.38 28.29 9.16 86.35 0.0133ENSG00000157110.11 RBPMS 135.68 51.42 17.47 188.47 0.01675ENSG00000114857.13 NKTR 137.91 92.83 23.91 169.09 0.0072ENSG00000197122.7 SRC 140.76 2.22 0.25 8.21 0.02595 ENSG00000163714.13U2SURP 151.90 162.07 42.15 384.12 0.0478 ENSG00000184465.11 WDR27 159.075.88 0.34 23.27 4.00E−04 ENSG00000118420.12 UBE3D 164.38 2.03 3.43 0.000.01815 ENSG00000078674.13 PCM1 168.30 78.81 20.62 277.38 0.0087ENSG00000227540.1 RP11-152N13.5 192.77 0.00 0.00 3.80 0.03325ENSG00000088448.10 ANKRD10 203.31 220.78 22.86 349.50 0.00545ENSG00000167508.6 MVD 206.88 29.59 1.56 103.48 0.0225 ENSG00000113013.8HSPA9 212.44 86.01 70.16 304.00 0.02625 ENSG00000111215.7 PRR4 216.81153.65 62.96 0.91 0.0136 ENSG00000101104.8 PABPC1L 217.65 0.20 6.83140.08 0.04645 ENSG00000108395.9 TRIM37 221.03 46.46 9.74 142.01 0.00515ENSG00000129932.3 DOHH 231.87 3.48 0.74 0.00 0.0136 ENSG00000091136.9LAMB1 235.00 123.61 46.18 317.48 0.0161 ENSG00000168000.10 BSCL2 262.44172.02 41.28 475.11 0.0411 ENSG00000103855.13 CD276 263.15 70.14 43.84307.87 0.0259 ENSG00000076242.10 MLH1 351.00 1.09 12.59 179.35 0.00955ENSG00000164292.8 RHOBTB3 360.56 48.32 128.06 23.71 0.0216ENSG00000132589.11 FLOT2 506.43 122.30 66.52 257.29 0.0479ENSG00000117523.11 PRRC2C 584.21 187.20 62.17 380.76 0.04055ENSG00000111642.10 CHD4 826.30 694.96 78.30 1169.69 0.0155ENSG00000100401.15 RANGAP1 896.77 106.57 36.05 257.91 0.03255ENSG00000141002.14 TCF25 1709.60 676.19 229.02 1118.74 0.03775ENSG00000198804.2 MT-CO1 2186.47 10593.20 1150.07 6626.71 0.0111ENSG00000101361.10 NOP56 2251.98 158.79 31.64 220.42 0.04485ENSG00000214548.10 MEG3 3738.76 600.30 96.15 384.80 0.02685

Example 8: CREB1 Affects Reprogramming Efficiency

In order to determine whether CREB1 plays a role in the reprogrammingprocess and thus affects reprogramming efficiency, overexpression andknockdown of CREB1 was performed using CREB1 overexpression (CREB1 OE)vectors and siRNA targeting CREB1 (siCREB1), respectively. DPSC1 andASC1 were either nucleofected with the CREB1 OE vectors or transfectedwith siCREB1 during reprogramming, then nucleofected with the episomalreprogramming factors. mRNA expression of CREB1 was significantlyincreased with CREB1 overexpression and significantly decreased withCREB1 knockdown in both DPSC and ASC lines (FIG. 7A). As shown in FIGS.7B and 7C, overexpression of CREB1 increased the reprogrammingefficiency in terms of the number of colonies and TRA-1-60-positivecells compared with the control (Scr CREB1) cells. Interestingly,knockdown of CREB1 drastically decreased the reprogramming efficiencyand on average only <1 iPS colony per well was generated (FIGS. 7B andC). Knockdown and overexpression of CREB1 in ASCs also showed similarresults. Knockdown of CREB1 significantly reduced the number of coloniesand overexpression of CREB1 significantly increased the number ofcolonies formed (FIG. 7C). These results showed that CREB1 expressionlevels significantly influence reprogramming efficiency, indicating animportant role of CREB1 in the reprogramming process into inducedpluripotency.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein. Other embodimentsare within the following claims. In addition, where features or aspectsof the invention are described in terms of Markush groups, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. Further, itwill be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention. Thecompositions, methods, procedures, treatments, molecules and specificcompounds described herein are presently representative of preferredembodiments are exemplary and are not intended as limitations on thescope of the invention. Changes therein and other uses will occur tothose skilled in the art which are encompassed within the spirit of theinvention are defined by the scope of the claims. The listing ordiscussion of a previously published document in this specificationshould not necessarily be taken as an acknowledgement that the documentis part of the state of the art or is common general knowledge.

The invention illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. The word “comprise” or variations such as“comprises” or “comprising” will accordingly be understood to imply theinclusion of a stated integer or groups of integers but not theexclusion of any other integer or group of integers. Additionally, theterms and expressions employed herein have been used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by exemplaryembodiments and optional features, modification and variation of theinventions embodied therein herein disclosed may be resorted to by thoseskilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention.

The content of all documents and patent documents cited herein isincorporated by reference in their entirety.

What is claimed is:
 1. A method of determining, in a sample, the presence and/or amount of pluripotent stem cells, said method comprising the steps of: (i) providing a sample suspected of containing one or more pluripotent stem cells; (ii) contacting the sample with a fluorescent compound under conditions that allow binding of said fluorescent compound to the pluripotent stem cells, wherein the fluorescent compound is a fluorescent compound of formula (I) or a pharmaceutically acceptable salt thereof

wherein: R₁, R₄, R₅, R₈, R₉ and R₁₀ are each independently selected from the group consisting of H and C₁₋₆ alkyl; R₂, R₃, R₆ and R₇ are each independently selected from C₁₋₆ alkyl; R₁₁ is selected from the group consisting of H, (CR₁₂R₁₃)_(o)—N(R₁₄R₁₅) and C₁₋₆ alkyl, wherein o is independently 0, 1, 2, 3, 4, or 5, and R₁₂-R₁₅ are each independently selected from the group consisting of H and C₁₋₆ alkyl; m and n are each independently 0, 1, or 2; p is 0, 1, 2, or 3, q is 1, 2, 3, or 4, with the proviso that p+q≤4; and

means that the respective bond can be a single or double bond and, if it is a single bond, the additional valencies are hydrogen; and (iii) determining the presence and/or amount of the pluripotent stem cells by measuring the fluorescence of the cells following said contacting.
 2. The method of claim 1, wherein the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.
 3. The method of claim 1, wherein the pluripotent stem cells are mammalian cells.
 4. The method of claim 1, wherein step (ii) does not comprise a washing step following said contacting.
 5. The method of claim 1 further comprising a step of: (iv) isolating the pluripotent stem cells labelled by the fluorescent compound from the sample.
 6. The method of claim 5, wherein the labelled cells are isolated by fluorescence-activated cell sorting (FACS).
 7. A method of determining, in a sample, the presence and/or amount of cells undergoing reprogramming to become induced pluripotent stem cells, said method comprising the steps of: (i) providing a sample suspected of containing one or more cells undergoing reprogramming to become induced pluripotent stem cells; (ii) contacting the sample with a fluorescent compound under conditions that allow binding of said fluorescent compound to the cells undergoing reprogramming to become induced pluripotent stem cells, wherein the fluorescent compound is a fluorescent compound of formula (I) or a pharmaceutically acceptable salt thereof

wherein: R₁, R₄, R₅, R₈, R₉ and R₁₀ are each independently selected from the group consisting of H and C₁₋₆ alkyl; R₂, R₃, R₆ and R₇ are each independently selected from C₁₋₆ alkyl; R₁₁ is selected from the group consisting of H, (CR₁₂R₁₃)_(o)—N(R₁₄R₁₅) and C₁₋₆ alkyl, wherein o is independently 0, 1, 2, 3, 4, or 5, and R₁₂-R₁₅ are each independently selected from the group consisting of H and C₁₋₆ alkyl; m and n are each independently 0, 1, or 2; p is 0, 1, 2, or 3, q is 1, 2, 3, or 4, with the proviso that p+q≤4; and

means that the respective bond can be a single or double bond and, if it is a single bond, the additional valencies are hydrogen; and (iii) determining the presence and/or amount of the cells undergoing reprogramming to become induced pluripotent stem cells by measuring the fluorescence of the cells following said contacting.
 8. The method of claim 7, wherein the cells undergoing reprogramming to become induced pluripotent stem cells are mammalian cells, preferably human, mouse, or rat cells, more preferably human cells.
 9. The method of claim 7, wherein step (ii) does not comprise a washing step following said contacting.
 10. The method of claim 7 further comprising a step of: (iv) isolating the cells undergoing reprogramming to become induced pluripotent stem cells labelled by the fluorescent compound from the sample.
 11. The method of claim 10, wherein the labelled cells are isolated by fluorescence-activated cell sorting (FACS). 